Modulation of survivin expression

ABSTRACT

Compounds and compositions are provided for modulating the expression of survivin. The compounds, exemplified by those acting through an RNAi antisense mechanism of action, include double-stranded and single-stranded constructs, as well as siRNAs, canonical siRNAs, blunt-ended siRNAs and single-stranded antisense RNA compounds. Methods of using these compounds for modulation of survivin expression and for treatment of diseases associated with expression of survivin are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of survivin. In particular, this invention relates toantisense compounds, particularly double-stranded oligonucleotides,specifically hybridizable with nucleic acids encoding human survivin.Such oligonucleotides have been shown to modulate the expression ofsurvivin.

BACKGROUND OF THE INVENTION

A hallmark feature of cancerous cells is uncontrolled proliferation.Among the differences that have been discovered between tumor and normalcells is resistance to the process of programmed cell death, also knownas apoptosis (Ambrosini et al., Nat. Med., 1997, 3, 917-921). Apoptosisis a process multicellular organisms have evolved to preventuncontrolled cell proliferation as well as to eliminate cells that havebecome sick, deleterious, or are no longer necessary. The process ofapoptosis involves a multistep cascade in which cells are degraded fromwithin through the concerted action of proteolytic enzymes and DNAendonucleases, resulting in the formation of apoptotic bodies that arethen removed by scavenger cells. Research to date has shown that much ofthe intracellular degradation is carried out through the action of thecaspases, a family of proteolytic enzymes that cleave adjacent toaspartate residues (Cohen, Biochemistry Journal, 1997, 326, 1-16).

The finding that most tumor cells display resistance to the apoptoticprocess has led to the view that therapeutic strategies aimed atattenuating the resistance of tumor cells to apoptosis could represent anovel means to halt the spread of neoplastic cells (Ambrosini et al.,Nat. Med., 1997, 3, 917-921). One of the mechanisms through which tumorcells are believed to acquire resistance to apoptosis is byoverexpression of survivin, a recently described member of the LAP(inhibitor of apoptosis) caspase inhibitor family. To date,overexpression of survivin has been detected in tumors of the lung,colon, pancreas, prostate, breast, stomach, non-Hodgkin's lymphoma, andneuroblastoma (Adida et al., Lancet, 1998, 351, 882-883; Ambrosini etal., Nat. Med., 1997, 3, 917-921; Lu et al., Cancer Res., 1998, 58,1808-1812). A more detailed analysis has been performed in neuroblastomawhere it was found that survivin overexpression segregated with tumorhistologies known to associate with poor prognosis (Adida et al.,Lancet, 1998, 351, 882-883). Finally, Ambrosini et al. describetransfection of HeLa cells with an expression vector containing a 708 ntfragment of the human cDNA encoding effector cell protease receptor 1(EPR-1), the coding sequence of which is complementary to the codingstrand of survivin (Ambrosini et al., J. Bio. Chem., 1998, 273,11177-11182). This construct caused a reduction in cell viability.

Survivin has recently been found to play a role in cell cycleregulation. It has been found to be expressed in the G2/M phase of thecell cycle in a cycle-regulated manner, and associates with microtubulesof the mitotic spindle. Disruption of this interaction results in lossof survivin's anti-apoptotic function and increased caspase-3 activityduring mitosis. Caspase-3 is associated with apoptotic cell death. It istherefore believed that survivin may counteract a default induction ofapoptosis in G2/M phase. It is believed that the overexpression ofsurvivin in cancer may overcome this apoptotic checkpoint, allowingundesired survival and division of cancerous cells. The survivinantisense construct described by Ambrosini above was found todownregulate endogenous survivin in HeLa cells and to increasecaspase-3-dependent apoptosis in cells in G2/M phase. Li et al., Nature,1998, 396, 580-584.

In many species, introduction of double-stranded RNA (dsRNA) inducespotent and specific gene silencing. This phenomenon occurs in bothplants and animals and has roles in viral defense and transposonsilencing mechanisms. (Jorgensen et al., Plant Mol. Biol., 1996, 31,957-973; Napoli et al., Plant Cell, 1990, 2, 279-289).

The first evidence that dsRNA could lead to gene silencing in animalscame from work in the nematode, Caenorhabditis elegans, where it hasbeen shown that feeding, soaking or injecting dsRNA (a mixture of bothsense and antisense strands) results in much more efficient silencingthan injection of either the sense or the antisense strands alone (Guoand Kemphues, Cell, 1995, 81, 611-620; Fire et al., Nature 391:806-811(1998); Montgomery et al., Proc. Natl. Acad. Sci. USA 95:15502-15507(1998); PCT International Publication W099/32619; (Fire et al., Nature,1998, 391, 806-810; Timmons et al., Gene, 2001, 263, 103-112; Timmonsand Fire, Nature, 1998, 395, 854). Since, the phenomenon has beendemonstrated in a number of organisms, including Drosophila melanogaster(Kennerdell et al., Cell 95:1017-1026 (1998)); and embryonic mice(Wianny et al., Nat. Cell Biol. 2:70-75 (2000)).

This posttranscriptional gene silencing phenomenon has been termed “RNAinterference” (RNAi) and has come to generally refer to the process ofgene silencing involving dsRNA which leads to the sequence-specificreduction of gene expression via target mRNA degradation (Tuschl et al.,Genes Dev., 1999, 13, 3191-3197).

It has been demonstrated that 21- and 22-nt dsRNA fragments having 3′overhangs are the canonical sequence-specific mediators of RNAi. Thesefragments, which are termed short interfering RNAs (siRNAs), aregenerated by an RNase III-like processing reaction from longer dsRNA.Chemically synthesized siRNA also mediate efficient target RNA cleavagewith the site of cleavage located near the center of the region spannedby the guiding strand of the siRNA. (Elbashir et al., Nature, 2001, 411,494-498). Characterization of the suppression of expression ofendogenous and heterologous genes caused by the 21-23 nucleotide siRNAshas been investigated in several mammalian cell lines, including humanembryonic kidney (293) and HeLa cells (Elbashir et al., Genes andDevelopment, 2001, 15, 188-200).

Recently, it has been shown that single-stranded RNA oligomers (ssRNAior asRNA) of antisense polarity can be potent inducers of gene silencingand that single-stranded oligomers are ultimately responsible for theRNAi phenomenon (Tijsterman et al., Science, 2002, 295, 694-697).

U.S. Pat. Nos. 5,898,031 and 6,107,094, each of which is hereinincorporated by reference, describe certain oligonucleotides havingRNA-like properties. When hybridized with RNA, these oligonucleotidesserve as substrates for a dsRNase enzyme with resultant cleavage of theRNA by the enzyme (Crooke, 2000; Crooke, 1999).

As a result of these advances in the understanding of apoptosis and therole that survivin expression is believed to play in conferring a growthadvantage to a wide variety of tumor cell types, there is a great desireto provide compositions of matter which can modulate the expression ofsurvivin. It is greatly desired to provide methods of diagnosis anddetection of nucleic acids encoding survivin in animals. It is alsodesired to provide methods of diagnosis and treatment of conditionsarising from survivin expression. In addition, improved research kitsand reagents for detection and study of nucleic acids encoding survivinare desired. Thus, the present invention provides a class of novelinhibitors of survivin, compositions comprising these compounds, andmethods of using the compounds.

SUMMARY OF THE INVENTION

The present invention is directed to oligomeric compounds, particularlysingle and double-stranded antisense compounds, which are targeted to anucleic acid encoding survivin, and which modulate the expression ofsurvivin. In some embodiments the antisense compounds areoligonucleotides. In some embodiments, the oligonucleotides are RNAioligonucleotides (which are predominantly RNA or RNA-like) Ii otherembodiments, the oligonucleotides are RNase H oligonucleotides (whichare predominantly DNA or DNA-like). In still other embodiments, theoligonucleotides may be chemically modified. Pharmaceutical and othercompositions comprising the antisense compounds of the invention arealso provided. Further provided are methods of modulating the expressionof survivin in cells or tissues comprising contacting said cells ortissues with one or more of the antisense compounds or compositions ofthe invention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of survivin by administering atherapeutically or prophylactically effective amount of one or more ofthe oligonucleotides or compositions of the invention. In anotherembodiment, the present invention provides for the use of a compound ofthe invention in the manufacture of a medicament for the treatment ofany and all conditions disclosed herein.

The disease or condition can be a hyperproliferative condition. In oneembodiment, the hyperproliferative condition is cancer.

The oligomeric compounds of the present invention are inhibitors ofsurvivin expression or overexpression. Because these compounds inhibitthe effects of survivin expression or overexpression, the compounds areuseful in the treatment of disorders related to survivin activity. Thus,the compounds of the present invention are antineoplastic agents.

The present compounds are believed to be useful in treating carcinomassuch as neoplasms of the central nervous system: glioblastomamultiforme, astrocytoma, oligodendroglial tumors, ependymal and choroidplexus tumors, pineal tumors, neuronal tumors, medulloblastoma,schwannoma, meningioma, meningeal sarcoma; neoplasms of the eye: basalcell carcinoma, squamous cell carcinoma, melanoma, rhabdomyosarcoma,retinoblastoma; neoplasms of the endocrine glands: pituitary neoplasms,neoplasms of the thyroid, neoplasms of the adrenal cortex, neoplasms ofthe neuroendocrine system, neoplasms of the gastroenteropancreaticendocrine system, neoplasms of the gonads; neoplasms of the head andneck: head and neck cancer, oral cavity, pharynx, larynx, odontogenictumors; neoplasms of the thorax: large cell lung carcinoma, small celllung carcinoma, non-small cell lung carcinoma, neoplasms of the thorax,malignant mesothelioma, thymomas, primary germ cell tumors of thethorax; neoplasms of the alimentary canal: neoplasms of the esophagus,neoplasms of the stomach, neoplasms of the liver, neoplasms of thegallbladder, neoplasms of the exocrine pancreas, neoplasms of the smallintestine, veriform appendix and peritoneum, adneocarcinoma of the colonand rectum, neoplasms of the anus; neoplasms of the genitourinary tract:renal cell carcinoma, neoplasms of the renal pelvis and ureter,neoplasms of the bladder, neoplasms of the urethra, neoplasms of theprostate, neoplasms of the penis, neoplasms of the testis; neoplasms ofthe female reproductive organs: neoplasms of the vulva and vagina,neoplasms of the cervix, addenocarcinoma of the uterine corpus, ovariancancer, gynecologic sarcomas; neoplasms of the breast; neoplasms of theskin: basal cell carcinoma, squamous cell carcinoma,dermatofibrosarcoma, Merkel cell tumor; malignant melanoma; neoplasms ofthe bone and soft tissue: osteogenic sarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, primitive neuroectodermaltumor, angiosarcoma; neoplasms of the hematopoietic system:myelodysplastic sydromes, acute myeloid leukemia, chronic myeloidleukemia, acute lymphocytic leukemia, HTLV-1 and T-ellleukemia/lymphoma, chronic lymphocytic leukemia, hairy cell leukemia,Hodgkin's disease, non-Hodgkin's lymphomas, mast cell leukemia; andneoplasms of children: acute lymphoblastic leukemia, acute myelocyticleukemias, neuroblastoma, bone tumors, rhabdomyosarcoma, lymphomas,renal tumors.

Thus, in one embodiment, the present invention provides a method for thetreatment of susceptible neoplasms comprising: administering to ananimal, particularly a human, an effective amount of a single-stranded(ssRNA or ssRNAi or asRNA) or double-stranded (dsRNA or siRNA)oligonucleotide directed to survivin. The ssRNA or dsRNA oligonucleotidemay be modified or unmodified. That is, the present invention providesfor the use of a double-stranded RNA oligonucleotide targeted tosurvivin, or a pharmaceutical composition thereof, for the treatment ofsusceptible neoplasms.

In another aspect, the present invention provides for the use of acompound of an isolated double-stranded RNA oligonucleotide in themanufacture of a medicament for inhibiting survivin expression oroverexpression. Thus, the present invention provides for the use of anisolated double-stranded RNA oligonucleotides targeted to survivin inthe manufacture of a medicament for the treatment of susceptibleneoplasms by means of the method described above.

The compounds of the present invention are especially useful for thetreatment of pancreatic cancer, prostate cancer, colon cancer, breastcancer, lung cancer, bladder cancer, liver cancer, ovarian cancer, renalcancer, glioblastoma, and non-Hodgkins lymphoma.

Another embodiment of the present invention is a method of treating ananimal, particularly a human, having a disease or conditioncharacterized by a reduction in apoptosis comprising administering tothe patient a therapeutically effective amount an of antisense compound8 to 80 nucleobases in length targeted to a nucleic acid moleculeencoding human survivin so that expression of survivin is inhibited.

The present invention also provides a method of modulating apoptosis ina cell comprising contacting a cell with an antisense compound 8 to 80nucleobases in length targeted to a nucleic acid molecule encoding humansurvivin so that apoptosis is modulated.

Still another embodiment of the invention is a method of modulatingcytokinesis in a cell comprising contacting a cell with an artisensecompound 8 to 80 nucleobases in length targeted to a nucleic acidmolecule encoding human survivin so that cytokinesis is modulated.

The present invention also provides a method of modulating the cellcycle in a cell comprising contacting a cell with an antisense compound8 to 80 nucleobases in length targeted to a nucleic acid moleculeencoding human survivin so that the cell cycle is modulated.

In still another embodiment of the invention, there is provided a methodof inhibiting the proliferation of cells comprising contacting cellswith an effective amount of an antisense compound 8 to 80 nucleobases inlength targeted to a nucleic acid molecule encoding human survivin, sothat proliferation of the cells is inhibited. In one embodiment, thecells are cancer cells. The method may further comprise administering tothe patient a chemotherapeutic agent.

In yet another embodiment, there is provided a method of modulatingapoptosis of hyperproliferative cells comprising contacting the cellswith an effective amount of antisense compound 8 to 80 nucleobases inlength targeted to a nucleic acid molecule encoding human survivin, sothat apoptosis of cells is modulated. In one embodiment, the cells arehyperproliferative cells and apoptosis is enhanced by the antisensecompound. In another embodiment, the modulation of apoptosis issensitization to an apoptotic stimulus. In one embodiment, the apoptoticstimulus is a cytotoxic chemotherapeutic agent. The method may furthercomprise contacting the cells with a chemotherapeutic agent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs double and single-stranded oligomericantisense compounds, particularly single or double-strandedoligonucleotides which are RNA or RNA-like and single-strandedoligonucleotides which are DNA or DNA-like for use in modulating thefunction of nucleic acid molecules encoding survivin, ultimatelymodulating the amount of survivin produced. This is accomplished byproviding antisense compounds which specifically hybridize with one ormore nucleic acids encoding survivin. As used herein, the terms “targetnucleic acid” and “nucleic acid encoding survivin” encompass DNAencoding survivin, RNA (including pre-mRNA and mRNA or portions thereof)transcribed from such DNA, and also cDNA derived from such RNA. Thespecific hybridization of an oligomeric compound with its complementarysense-orientation target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense,” and such compounds can bedescribed as being in the “antisense orientation” relative to thetarget.

The functions of DNA to be interfered with include replication andtranscription. The functions of RNA to be interfered with include, forexample, translocation of the RNA to the site of protein translation,translation of protein form the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression of survivinat the RNA and/or the protein level. In the context of the presentinvention, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression. In the context of the presentinvention, inhibition is a desired form of modulation of gene expressionand RNA, and in some embodiments mRNA, is a suitable target.

It is suitable to target specific nucleic acids for antisense;“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding survivin. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the mRNA and or protein, will result. Withinthe context of the present invention, one intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG and 5′-CUG,while the translation initiation codons 5′-AUA, 5′-ACG and 5′-CUG havebeen shown to function in vivo. Thus, the terms “translation initiationcodon” and “start codon” can encompass many codon sequences, even thoughthe initiator amino and in each instance is typically methionine (ineukaryotes) or formylmethionine (in prokaryotes). It is also known inthe art that eukaryotic and prokaryotic genes may have two or morealternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAmolecule transcribed from a gene encoding survivin, regardless of thesequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-TAA,5′-TAG or 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively). The terms “start codon region” and “translationinitiation codon region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3) from a translation initiation codon.Similarly, the terms “stop codon region” and “translation terminationcodon region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation termination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be an effective targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be target regions, and are particularly useful insituations where aberrant splicing is implicated in disease, or where anoverproduction of a particular mRNA splice product is implicated indisease.

Once one or more target sites have been identified, antisense oligomericcompounds, typically an antisense oligonucleotide, are chosen which aresufficiently complementary to the target, i.e., hybridize sufficientlywell and with sufficient specificity, to give the desired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a complete orpartial loss of function, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of therapeutictreatment, or under conditions in which in vitro or in vivo assays areperformed. Moreover, an oligonucleotide may hybridize over one or moresegments such that intervening or adjacent segments are not involved inthe hybridization event (e.g., a loop structure, mismatch or hairpinstructure). The compounds of the present invention comprise at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99°/, or 100% sequence complementarity to a target regionwithin the target nucleic acid sequence to which they are targeted. Forexample, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention.

Percent complementarity of an antisense compound with a region of atarget nucleic acid can be determined routinely using BLAST programs(basic local alignment search tools) and PowerBLAST programs known inthe art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang andMadden, Genome Res., 1997, 7, 649-656). Percent homology, sequenceidentity or complementarity, can be determined by, for sample, the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, Madison Wis.), usingdefault settings, which uses the algorithm of Smith and Waterman (Adv.Appl. Math., 1981, 2, 482-489). In some embodiments, homology sequenceidentity or complementarity, between the antisense compound and targetis between about 50% to about 60%. In some embodiments, homology,sequence identity or complementarity, is between about 60% to about 70%.In some embodiments, homology, sequence identity or complementarity, isbetween about 70% and about 80%. In some embodiments, homology, sequenceidentity or complementarity, is between about 80% and about 90%. In someembodiments, homology, sequence identity or complementarity, is about90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99% or about 100%.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

Multiple mechanisms exist by which short synthetic oligonucleotides canbe used to modulate gene expression in mammalian cells. A commonlyexploited antisense mechanism is RNase H-dependent degradation of thetargeted RNA. RNase H is a ubiquitously expressed endonuclease thatrecognizes antisense DNA-RNA heteroduplex, hydrolyzing the RNA strand. Afurther antisense mechanism involves the utilization of enzymes thatcatalyze the cleavage of RNA-RNA duplexes. These reactions are catalyzedby a class of RNAse enzymes including but not limited to RNAse III andRNAse L. The antisense mechanism known RNA interference (RNAi) isoperative on RNA-RNA hybrids and the like.

Both RNase H-based antisense (usually using single-stranded compounds)and RNA interference (usually using double-stranded compounds known assiRNAs) are antisense mechanisms, typically resulting in loss of targetRNA function.

Optimized siRNA and RNase H-dependent oligomeric compounds behavesimilarly in terms of potency, maximal effects, specificity and durationof action, and efficiency. Moreover it has been shown that in general,activity of dsRNA constructs correlated with the activity of RNaseH-dependent single-stranded antisense compounds targeted to the samesite. One major exception is that RNase H-dependent antisense compoundswere generally active against target sites in pre-mRNA whereas siRNAswere not.

These data suggest that, in general, sites on the target RNA that werenot active with RNase H-dependent oligonucleotides were similarly notgood sites for siRNA. Conversely, a significant degree of correlationbetween active RNase H oligonucleotides and siRNA was found, suggestingthat if a site is available for hybridization to an RNase Holigonucleotide, then it is also available for hybridization andcleavage by the siRNA complex. Consequently, once suitable target siteshave been determined by either antisense approach, these sites can beused to design constructs that operate by the alternative antisensemechanism (Vickers et al., 2003, J. Biol. Chem. 278, 7108). Moreover,once a site has been demonstrated as active for either an RNAi or anRNAse H oligonucleotide, a single-stranded RNAi oligonucleotide (ssRNAior asRNA) can be designed.

In some embodiments of the present invention, double-stranded antisenseoligonucleotides are suitable. These double-stranded antisenseoligonucleotides may be RNA or RNA-like, and may be modified orunmodified, in that the oligonucleotide, if modified, retains theproperties of forming an RNA:RNA hybrid and recruitment and (activation)of a dsRNase. In other embodiments, the single-stranded oligonucleotides(ssRNAi or asRNA) may be RNA-like.

In other embodiments of the present invention, single-stranded antisenseoligonucleotides are suitable. In some embodiments, the single-strandedoligonucleotides may be “DNA-like”, in that the oligonucleotide has wellcharacterized structural features, for example a plurality of unmodified2′ Hs or a stabilized backbone such as e.g., phosphorothioate, that isstructurally suited for interaction with a target oligonucleotide andrecruitment and (activation) of RNase H.

While oligonucleotides are one form of antisense compound, the presentinvention comprehends other oligomeric antisense compounds, includingbut not limited to oligonucleotide mimetics such as are described below.The compounds in accordance with this invention can comprise from about8 to about 80 nucleobases. In another embodiment, the oligonucleotide isabout 10 to 50 nucleotides in length. In yet another embodiment, theoligonucleotide is 12 to 30 nucleotides in length. In yet anotherembodiment, the oligonucleotide is 12 to 24 nucleotides in length. Inyet another embodiment, the oligonucleotide is 19 to 23 nucleotides inlength. Some embodiments comprise at least an 8-nucleobase portion of asequence of an oligomeric compound which inhibits expression ofsurvivin. dsRNA or siRNA molecules directed to survivin, and their usein inhibiting survivin mRNA expression, are also embodiments within thescope of the present invention.

The oligonucleotides of the present invention also include variants inwhich a different base is present at one or more of the nucleotidepositions in the oligonucleotide. For example, if the first nucleotideis an adenosine, variants may be produced which contain thymidine (oruridine if RNA), guanosine or cytidine at this position. This may bedone at any of the positions of the oligonucleotide. Thus, a 20-mer maycomprise 60 variations (20 positions×3 alternates at each position) inwhich the original nucleotide is substituted with any of the threealternate nucleotides. These oligonucleotides are then tested using themethods described herein to determine their ability to inhibitexpression of survivin mRNA.

Oligomeric Compounds

In the context of the present invention, the term “oligomeric compound”refers to a polymeric structure capable of hybridizing to a region of anucleic acid molecule. This term includes oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics andchimeric combinations of these, Oligomeric compounds are routinelyprepared linearly but can be joined or otherwise prepared to be circularand may also include branching. Oligomeric compounds can includedouble-stranded constructs such as, for example, two strands hybridizedto form double-stranded compounds or a single strand with sufficientself complementarity to allow for hybridization and formation of a fullyor partially double-stranded compound. In one embodiment of theinvention, double-stranded antisense compounds encompass shortinterfering RNAs (siRNAs). As used heroin, the term “siRNA” is definedas a double-stranded compound having a first and second strand andcomprises a central complementary portion between said first and secondstrands and terminal portions that are optionally complementary betweensaid first and second strands or with the target mRNA. Each strand maybe from about 8 to about 80 nucleobases in length, 10 to 50 nucleobasesin length, 12 to 30 nucleobases in length, 12 to 24 nucleobases inlength or 19 to 23 nucleobases in length. The central complementaryportion may be from about 8 to about 80 nucleobases in length, 10 to 50nucleobases in length, 12 to 30 nucleobases in length, 12 to 24nucleobases in length or 19 to 23 nucleobases in length. The terminalportions can be from 1 to 6 nucleobases in length. The siRNAs may alsohave no terminal portions. The two strands of an siRNA can be linkedinternally leaving free 3′ or 5′ termini or can be linked to form acontinuous hairpin structure or loop. The hairpin structure may containan overhang on either the 5′ or 3′ terminus producing an extension ofsingle-stranded character.

In one embodiment of the invention, double-stranded antisense compoundsare canonical siRNAs. As used herein, the term “canonical siRNA” isdefined as a double-stranded oligomeric compound having a first strandand a second strand each strand being 21 nucleobases in length with thestrands being complementary over 19 nucleobases and having on each 3′termini of each strand a deoxy thymidine dimer (dTdT) which in thedouble-stranded compound acts as a 3′ overhang.

In another embodiment, the double-stranded antisense compounds areblunt-ended siRNAs. As used herein the term “blunt-ended siRNA” isdefined as an siRNA having no terminal overhangs. That is, at least oneend of the double-stranded compound is blunt. siRNAs whether canonicalor blunt act to elicit dsRNAse enzymes and trigger the recruitment oractivation of the RNAi antisense mechanism. In a further embodiment,single-stranded RNAi (ssRNAi) compounds that act via the RNAi antisensemechanism are contemplated.

Further modifications can be made to the double-stranded compounds andmay include conjugate groups attached to one of the termini, selectednucleobase positions, sugar positions or to one of the internucleosidelinkages. Alternatively, the two strands can be linked via a non-nucleicacid moiety or linker group. When formed from only one strand, dsRNA cantake the form of a self-complementary hairpin-type molecule that doublesback on itself to form a duplex. Thus, the dsRNAs can be fully orpartially double-stranded. When formed from two strands, or a singlestrand that takes the form of a self-complementary hairpin-type moleculedoubled back on itself to form a duplex, the two strands (orduplex-forming regions of a single strand) are complementary RNA strandsthat base pair in Watson-Crick fashion.

In general an oligomeric compound comprises a backbone of momericsubunits joined linking groups where each linked momeric subunit isdirectly or indirectly attached to a heterocyclic base moiety.Oligomeric compounds may also include monomeric subunits that are notlinked to a heterocyclic base moiety thereby providing abasic sites. Anyone of the repeated units making up an oligomeric compound cm bemodified giving rise to a variety of motifs including hemimers, gapmersand chimeras.

As is known in the art, a nucleoside comprises a sugar moiety attachedto a heterocyclic base moiety. The two most common classes of suchheterocyclic bases are purines and pyrimidines. Nucleotides arenucleosides that further include a phosphate group covalently linked tothe sugar portion of the nucleoside. For those nucleosides that includea pentofuranosyl sugar, the phosphate group can be linked to either the2′, 3′ or 5′ hydroxyl moiety of the sugar giving the more common 3′,5-internucleoside linkage or the not so common 2′,5′-internucleosidelinkage. In forming oligonucleotides, the phosphate groups covalentlylink the sugar moieties of adjacent nucleosides. The respective ends canbe joined to form a circular structure by hybridization or by formationof a covalent bond. In addition, linear compounds may have internalnucleobase complementarity and may therefore fold in a manner as toproduce a fully or partially double-stranded compound. Withinoligonucleotides, the phosphate groups are commonly referred to asforming the internucleoside linkage or in conjunction with the sugarring form the backbone of the oligonucleotide. The normalinternucleoside linkage that comprises the backbone of RNA and DNA is a3′ to 5′ phosphodiester linkage. However, open linear structures aregenerally desired.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside linkages, as well as oligonucleotide analogs orchemically modified oligonucleotides that have one or more non-naturallyoccurring portions which function in a similar manner. Such modified orsubstituted oligonucleotides are suitable over the naturally occurringforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for a nucleic acid target andenhanced nuclease stability.

In the context of this invention, the term “oligonucleoside” refers to asequence of nucleosides that are joined by internucleoside linkages thatdo not have phosphorus atoms. Internucleoside linkages of this typeinclude short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixedheteroatom cycloalkyl, one or more short chain heteroatomic linkers andone or more short chain heterocyclic linkers. These internucleosidelinkages include but are not limited to siloxane, sulfide, sulfoxide,sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl,thioformacetyl, alken, sulfamate; methyleneimino, methylenehydrazino,sulfonate, sulfonamide, amide and others having mixed N, O, S and CH₂component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of whichis herein incorporated by reference.

Further included in the present invention are antisense oligomericcompounds including antisense oligonucleotides, external guide sequence(EGS) oligonucleotides, alternate splicers, and other oligomericcompounds which hybridize to at least a portion of the target nucleicacid. As such, these antisense oligomeric compounds may be introduced inthe form of single-stranded, double-stranded, circular or hairpinoligomeric compounds and may contain structural elements such asinternal or terminal bulges, mismatches or loops. In general, nucleicacids (including oligonucleotides) may be described as “DNA-like” (i.e.,having 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., having 2′-hydroxyl or 2′-modified sugars and,generally U rather than T bases). Once introduced to a system, theoligomeric compounds of the invention may elicit die action of one ormore enzymes or structural proteins to effect modification of the targetnucleic acid.

The oligomeric compounds in accordance with this invention can comprisefrom about 8 to about 80 nucleobases (i.e. from about 8 to about 80linked nucleobases and/or monomeric subunits). One of ordinary skill inthe art will appreciate that the invention embodies oligomeric compoundsof 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,or 80 nucleobases in length.

In one embodiment, the oligomeric compounds of the invention are 10 to50 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleobases in length.

In another embodiment, the oligomeric compounds of the invention are 12to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleobases in length.

In a further embodiment, the oligomeric compounds of the invention are12 to 24 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleobases in length.

In another embodiment, the oligomeric compounds of the invention are 19to 23 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 19, 20, 21, 22 or23 nucleobases in length.

One particularly length for oligomeric compounds is from about 12 toabout 30 nucleobases. Another particularly length is from about 12 toabout 24 nucleobases. A further particularly suitable length is fromabout 19 to about 23 nucleobases.

Chimeric Oligomeric Compounds

It is not necessary for all positions in a oligomeric compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligomeric compound oreven at a single monomeric subunit such as a nucleoside within aoligomeric compound. The present invention also includes oligomericcompounds which are chimeric oligomeric compounds. “Chimeric” oligomericcompounds or “chimeras,” in the context of this invention, areoligomeric compounds containing two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, alteration of charge, and/or increasedbinding affinity for the target nucleic acid. An additional region ofthe oligomeric compound may serve as a substrate for enzymes capable ofcleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is acellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby greatly enhancing the efficiency of inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligomeric compounds when chimeras are used, compared to forexample phosphorothioate deoxyoligonucleotides hybridizing to the sametarget region. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art. Similar observations are madefor chimeras that form RNA:RNA hybrids and are substrates for dsRNases.

Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotideanalogs, oligonucleosides and/or oligonucleotide mimetics as describedabove. Routinely used chimeric compounds include but are not limited tohybrids, hemimers, gapmers, inverted gapmers and blockmers wherein thevarious point modifications and or regions are selected from native ormodified DNA and RNA type units and or mimetic type subunits such ES forexample locked nucleic acids (LNA) (which encompasses ENA™ as describedbelow), peptide nucleic acids (PNA), morpholinos, and others. These aredescribed below. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, each of which is herein incorporated by reference in itsentirety.

Oligomer Mimetics

Another group of oligomeric compounds amenable to the present inventionincludes oligonucleotide mimetics. The term mimetic as it is applied tooligonucleotides is intended to include oligomeric compounds wherein thefuranose ring or the furanose ring and the internucleotide linkage arereplaced with novel groups, replacement of only the furanose ring isalso referred to in the art as being a sugar surrogate. The heterocyclicbase moiety or a modified heterocyclic base moiety is maintained forhybridization with an appropriate target nucleic acid.

One such oligomeric compound, an oligonucleotide mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). PNAs have favorable hybridizationproperties, high biological stability and are electrostatically neutralmolecules. In one recent study PNAs were used to correct aberrantsplicing in a transgenic mouse model (Sazani of al., Nat. Biotechnol.,2002, 20, 1228-1233). In PNA oligomeric compounds, the sugar-backbone ofan oligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are bounddirectly or indirectly (—C(—O)—CH₂— as shown below) to aza nitrogenatoms of the amide portion of the backbone. Representative United Statespatents that teach the preparation of PNA oligomeric compounds include,but are not limited to, U.S. Pat. Nos. 5,539,032; 5,714,331; and5,719,262, each of which is herein incorporated by reference. PNAs canbe obtained commercially from Applied Biosystems (Foster City, Calif.,USA).

Numerous modifications have been made to the basic PNA backbone since itwas introduced in 1991 by Nielsen and coworkers Nielsen et al., Science,1991, 254, 1497-1500). The basic structure is shown below.

wherein

Bx is a heterocyclic base moiety;

T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted orunsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the ω-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the α-aminogroup or optionally through the ω-amino group when the amino acid islysine or ornithine or a peptide derived from D, L or mixed D and Lamino acids linked through an amino group; a chemical functional group,a reporter group or a conjugate group;

Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

Z₂ is hydrogen, C₁-C₆ allyl, an amino protecting group,—C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxylgroup or optionally through the ω-carboxyl group when the amino acid isaspartic acid or glutamic acid or a peptide derived from D, L or mixed Dand L amino acids linked through a carboxyl group;

Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃,benzyl, benzoyl, or —(CH₂), —N(H)Z₁;

each J is O, S or NH;

R₅ is a carbonyl protecting group; and

n is from 7 to about 79.

Another class of oligonucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. One class of linking groups have been selected to give anon-ionic oligomeric compound. The non-ionic morpholino-based oligomericcompounds are less likely to have undesired interactions with cellularproteins. Morpholino-based oligomeric compounds are non-ionic mimics ofoligonucleotides which are less likely to form undesired interactionswith cellular proteins (Dwaine A. Braasch and David R. Corey,Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomericcompounds have been studied in zebrafish embryos (see: Genesis, volume30, issue 3, 2001 and Heasman, J., Dev. Biol., 2002, 243, 209-214).Further studies of morpholino-based oligomeric compounds have also beenreported (see: Nasevicius et al., Nat. Genet., 2000, 26, 216-220; andLacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596).Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No.5,034,506, issued Jul. 23, 1991. The morpholino class of oligomericcompounds have been prepared having a variety of different linkinggroups joining the monomeric subunits.

Morpholino nucleic acids have been prepared having a variety ofdifferent linking groups (L₂) joining the monomeric subunits. The basicformula is shown below:

wherein

T₁ is hydrogen, hydroxyl, a protected hydroxyl, a linked nucleoside or alinked oligomeric compound;

T₅ is hydrogen or a phosphate, phosphate derivative, a linked nucleosideor a linked oligomeric compound; and

L₂ is a linking group which can be varied from chiral to achiral fromcharged to neutral (U.S. Pat. No. 5,166,315 discloses linkages including—O—P(═O)(N(CH₃)₂)—O—; U.S. Pat. No. 5,034,506 discloses achiralintermorpholino linkages such as for example: —S(═O)—X— where X is NH,NCH₃, O, S, or CH₂; —C(═Y)—O— where Y is O or S; —S(═O)(OH)—C₂—;—S(═O)(OH)—N(R)—CH₂— where R is H or CH₃; and U.S. Pat. No. 5,185,444discloses phosphorus containing chiral intermorpholino linkages such asfor example: —P(═O)(—X)—O— where X is F, CH₂R, S—CH₂R or NR₁R₂ and eachR, R₁ and R₂ is H, CH₃ or some other moiety that doesn't interfere withthe base specific hydrogen bonding; and

n is from 7 to about 79.

A further class of oligonucleotide mimetic is referred to ascyclohexenyl nucleic acids (CeNA). The furanose ring normally present inan DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMTprotected phosphoramidite monomers have been prepared and used foroligomeric compound synthesis following classical phosphoramiditechemistry. Fully modified CeNA oligomeric compounds and oligonucleotideshaving specific positions modified with CeNA have been prepared andstudied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Ingeneral the incorporation of CeNA monomers into a DNA chain increasesits stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexeswith RNA and DNA complements with similar stability to the nativecomplexes. The study of incorporating CeNA structures into naturalnucleic acid structures was shown by NMR and circular dichroism toproceed with easy conformational adaptation. Furthermore theincorporation of CeNA into a sequence targeting RNA was stable to serumand able to activate E. Coli RNase resulting in cleavage of the targetRNA strand.

The general formula of CeNA is shown below;

wherein

each Bx is a heterocyclic base moiety;

L₃ is an inter cyclohexenyl linkage such as for example a phosphodiesteror a phosphorothioate linkage;

T₁ is hydrogen, hydroxyl, a protected hydroxyl, a linked nucleoside or alinked oligomeric compound; and

T₂ is hydrogen or a phosphate, phosphate derivative, a linked nucleosideor a linked oligomeric compound.

Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid)can be prepared from one or more anhydrohexitol nucleosides (see,Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) andwould have the general formula:

each Bx is a heterocyclic base moiety;

L is an inter anhydrohexitol linkage such as for example aphosphodiester or a phosphorothioate linkage;

T₁ is hydrogen, hydroxyl, a protected hydroxyl, a linked nucleoside or alinked oligomeric compound; and

T₂ is hydrogen or a phosphate, phosphate derivative, a linked nucleosideor a linked oligomeric compound.

A further modification includes bicyclic sugar moieties such as “LockedNucleic Acids” (LNAs) in which the 2′-hydroxyl group of the ribosylsugar ring is linked to the 4′ carbon atom of the sugar ring therebyforming a 2′-C,4′-C-oxymethylene linkage to form the bicyclic sugarmoiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001,2, 558-561; Braasch et al., Chem. Biol., 2001, 81-7; and Orum et al.,Curr. Opinion Ther. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos.6,268,490 and 6,670,461). The linkage can be a (—CH₂—)x group bridgingthe 2′ oxygen atom and the 4′ carbon atom, wherein if x=1 the term LNAis used, if x=2 the term ENA™ is used (Singh et al., Chem. Commun.,1998, 4, 455-456; ENA™: Morita et al., Bioorganic Medicinal Chemistry,2003, 11, 2211-2226). Thus, “ENA™” is one non limiting example of anLNA. LNA and other bicyclic sugar analogs display very high duplexthermal stabilities with complementary DNA and RNA (Tm=+3 to +10 C),stability towards 3′-exonucleolytic degradation and good solubilityproperties. LNAs are commercially available from ProLigo (Paris, Franceand Boulder, Colo., USA). The basic structure of an LNA having a singleCH₂ linkage in the bicyclic ring system is shown below. This is merelyillustrative of one type of LNA.

wherein each T₁ and T₂ is, independently, hydrogen, a hydroxylprotecting group, a linked nucleoside or a linked oligomeric compound,and each Z, is an internucleoside linking group such as for examplephosphodiester or phosphorothioate.

An isomer of LNA that has also been studied is alpha-L-LNA which hasbeen shown to have superior stability against a 3′-exonuclease (Friedenet al., Nucleic Acids Research, 2003, 21, 6365-6372). The alpha-L-LNA'swere incorporated into antisense gapmers and chimeras that showed potentantisense activity. The structure of alpha-L-LNA is shown below:

Another similar bicyclic sugar moiety that has been prepared and studiedhas the bridge going from the 3′-hydroxyl group via a single methylenegroup to the 4′ carbon atom of the sugar ring thereby forming a3′-C,4′-C-oxymethylene linkage (see U.S. Pat. No. 6,043,060).

The conformations of LNAs determined by 2D NMR spectroscopy have shownthat the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of three (3) LNA monomers(T or A) significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination ofan LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.DN/LNA chimeras have been shown to efficiently inhibit gene expressionwhen targeted to a variety of regions (5′-untranslated region, region ofthe start codon or coding region) within the luciferase mRNA (Braasch etal., Nucleic Acids Research, 2002, 30, 5160-5167).

Novel types of LNA-oligomeric compounds, as well as the LNAs, are usefulin a wide range of diagnostic and therapeutic applications. Among theseare antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide-based drugs.

Potent and nontoxic antisense oligonucleotides containing LNAs have beendescribed (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97,5633-5638.) The authors have demonstrated that LNAs confer severaldesired properties to antisense compounds. LNA/DNA copolymers were notdegraded readily in blood serum and cell extracts. LNA/DNA copolymersexhibited potent antisense activity in assay systems as disparate asG-protein-coupled receptor signaling in living rat brain and detectionof reporter genes in Escherichia coli. Lipofectin-mediated efficientdelivery of LNA into living human breast cancer cells has also beenaccomplished. Further successful in vivo studies involving LNA's haveshown knock-down of the rat delta opioid receptor without toxicity(Wahlestedl et al., Proc. Natl. Acad. Sot., 2000, 97, 5633-5638) and inanother study showed a blockage of the translation of the large subunitof RNA polymerase II (Fluiter et al., Nucleic Acids Res., 2003, 31,953-962).

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, havealso been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO03/020739; and WO99/14226). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-amino- and 2′-methylamino-LNA's have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

Another oligonucleotide mimetic amenable to the present invention thathas been prepared and studied is threose nucleic acid. Thisoligonucleotide mimetic is based on threose nucleosides instead ofribose nucleosides and has the general structure shown below:

Initial interest in (3′,2′)-alpha-L-threose nucleic acid (TNA) wasdirected to the question of whether a DNA polymerase existed that wouldcopy the TNA. It was found that certain DNA polymerases are able to copylimited stretches of a TNA template (reported in C&EN/Jan. 13, 2003).

In another study it was determined that TNA is capable of antiparallelWatson-Crick base pairing with complementary DNA, RNA and TNAoligonucleotides (Chaput et al., J. Am. Chem. Soc., 2003, 125, 856-857).

In one study (3′,2′)-alpha-L-threose nucleic acid was prepared andcompared to the 2′ and 3′ amidate analogs (Wu et al., Organic Letters,2002, 4(8), 1279-1282). The amidate analogs were shown to bind to RNAand DNA with comparable strength to that of RNA/DNA.

Further oligonucleotide mimetics have been prepared to include bicyclicand tricyclic nucleoside analogs having the formulas (amidite monomersshown):

(sec Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens etal., J. Am. Chem. Soc., 1999, 121, 3249-3255; Renneberg et al., J. Am.Chem. Soc., 2002, 124, 5993-6062; and Renneberg et al., Nucleic acidsres., 2002, 30, 2751-2757). These modified nucleoside analogs have beenoligomerized using the phosphoramidite approach and the resultingoligomeric compounds containing tricyclic nucleoside analogs have shownincreased thermal stabilities (Tm's) when hybridized to DNA, RNA anditself. Oligomeric compounds containing bicyclic nucleoside analogs haveshown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acids which incorporate a phosphorus group inthe backbone. This class of olignucleotide mimetic is reported to haveuseful physical and biological and pharmacological properties in theareas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

The general formula (for definitions or Markush variables see: U.S. Pat.Nos. 5,874,553 and 6,127,346 herein incorporated by reference in theirentirety) is shown below.

Further oligonucleotide mimetics amenable to the present invention havebeen prepared wherein a cyclobutyl ring replaces the naturally occurringfuranosyl ring.

Modified Internucleoside Linkages

Specific examples of antisense oligomeric compounds useful in thisinvention include oligonucleotides containing modified e.g.non-naturally occurring internucleoside linkages. As defined in thisspecification, oligonucleotides having modified internucleoside linkagesinclude internucleoside linkages that retain a phosphorus atom andinternucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

Modified oligonucleotide backbones containing a phosphorus atom thereininclude, for example, phosphotothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoranidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, phosphonoacetateand thiophosphonoacetate (see Sheehan et al., Nucleic Acids Research,2003, 31(14), 4109-4118 and Dellinger et al., J. Am. Chem. Soc., 2003,125, 940-950), selenophosphates and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′to 5′ or 2′ to 2′ linkage. One phosphorus containing modifiedinternucleoside linkage is the phosphorothioate internucleoside linkage,which is linked in a 3′-5′ linkage. Oligonucleotides having invertedpolarity comprise a single 3′ to 3′ linkage at the 3′-mostinternucleotide linkage i.e. a single inverted nucleoside residue whichmay be abasic (the nucleobase is missing or has a hydroxyl group inplace thereof). Various salts, mixed salts and free acid forms are alsoincluded.

N3′-P5′-phosphoramidates have been reported to exhibit both a highaffinity towards a complementary RNA strand and nuclease resistance(Gryaznov et al., J. Am. Chem. Soc., 1994, 116, 3143-3144).N3′-P5′-phosphoramidates have been studied with some success in vivo tospecifically down regulate the expression of the c-myc gene (Skorski etal., Proc. Natl. Acad. Sci., 1997, 94, 3966-3971; and Faira et al., Nat.Biotechnol., 2001, 19, 40-44).

Representative United States patents that teach the preparation of theabove phosphorus containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, each of which is herein incorporated by reference.

In some embodiments of the invention, oligomeric compounds have one ormore phosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). The MMI type internucleoside linkages aredisclosed in the above referenced U.S. Pat. No. 5,489,677. Amideinternucleoside linkages are disclosed in the above referenced U.S. Pat.No. 5,602,240.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of whichis herein incorporated by reference.

Modified Sugars

Oligomeric compounds of the invention may also contain one or moresubstituted or other wise modified sugar moieties. Ribosyl and relatedsugar moieties are routinely modified at any reactive position notinvolved in linking. Thus a suitable position for a sugar substituentgroup is the 2′-position not usually used in the native 3′ to5′-internucleoside linkage. Other suitable positions are the 3′ and the5′-termini. 3′-sugar positions are open to modification when the linkagebetween two adjacent sugar units is a 2′,5′-linkage. Sugar substituentgroups include: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly suitable are O((CH₂)_(n)O)_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from 1 to about 10. Othersuitable oligonucleotides comprise a sugar substituent group selectedfrom: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties.

One modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also knownas 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta,1995, 78, 486-504) i.e., an alkoxyalkoxy group. Further modificationsincludes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, alsoknown as 2′-DMAOE, as described in examples hereinbelow,2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—(CH₂)₂O—(CH₂)₂N(CH₃, and N-methylacetamide (also referred to asNMA, 2′-O—CH₂—C(═O)—N(H)CH₃.)

Other sugar substituent groups include methoxy (—O—CH₃), aminopropoxy(—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) andfluoro (F). 2′-Sugar substituent groups may be in the arabino (up)position or ribo (down) position. One 2′-arabino modification is 2′-F(see: Loc et al., Biochemistry, 2002, 41, 3457-3467). Similarmodifications may also be made at other positions on the oligomericcompound, particularly the 3′ position of the sugar on the 3′ terminalnucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′terminal nucleotide. Oligomeric compounds may also have sugar mimeticssuch as cyclobutyl moieties in place of the pentofuranosyl sugar.Representative United States patents that teach the preparation of suchmodified sugar structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; 5,792,747; 5,700,920; and 6,147,200 each of which is hereinincorporated by reference in its entirety.

Further representative sugar substituent groups include groups offormula I_(a) or II_(a):

wherein:

R_(b) is O, S or NH;

R_(d) is a single bond, O, S or C(═O);

R_(c) s C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)), or has formula III_(a):

R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;

R_(r) is —R_(x)-R_(y);

each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or aconjugate group, wherein the substituent groups are selected fromhydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R_(u) and R_(v), together form a phthalimido moiety withthe nitrogen atom to which they are attached;

each R_(w) is, independently, substituted or unsubstituted C₁-C₁₀ alkyl,trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy,benzyloxy, butyl, iso-butyryl, phenyl or aryl;

R_(k) is hydrogen, a nitrogen protecting group or —R_(x)-R_(y);

R_(x) is a bond or a linking moiety;

R_(y) is a chemical functional group, a conjugate group or a solidsupport medium;

each R_(m) and R_(n) is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein thesubstituent groups are selected from hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl,alkynyl; NH₃ ⁺, N(R_(u))(R_(v)), guanidino and acyl where said acyl isan acid amide or an ester;

or R_(k) and R_(m), together, are a nitrogen protecting group, arejoined in a ring structure that optionally includes an additionalheteroatom selected from N and O or are a chemical functional group;

R_(i) is OR_(z), SR_(z), or N(R_(z))₂;

each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(m)R_(u), C(—O)N(H)R_(u) or OC(—O)N(H)R_(u);

R_(f), R_(g) and R_(h) comprise a ring system having from about 4 toabout 7 carbon atoms or having from about 3 to about 6 carbon atoms and1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(R_(k))(R_(m))OR_(k),halo, SR_(k) or CN;

ma is 1 to about 10;

each mb is, independently, 0 or 1;

me is 0 or an integer from 1 to 10;

md is an integer from 1 to 10;

me is from 0, 1 or 2; and

provided that when mc is 0, md is greater than 1.

Representative substituents groups of Formula I are disclosed in U.S.Pat. No. 6,172,209, entitled “Capped 2′-Oxyethoxy Oligonucleotides,”hereby incorporated by reference in its entirety.

Representative cyclic substituent groups of Formula II are disclosed inU.S. Pat. No. 6,271,358, entitled “RNA Targeted 2′-Oligomeric compoundsthat are Conformationally Preorganized,” hereby incorporated byreference in its entirety.

Sugar substituent groups include O((CH₂)_(n)O)_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂ andO(CH₂)_(n)ON((CH₂)_(n)CH₃))₂, where n and m are from 1 to about 10.

Representative guanidino substituent groups that are shown in formulaIII are disclosed in U.S. Pat. No. 6,593,466, entitled “FunctionalizedOligomers”, filed Jul. 7, 1999, hereby incorporated by reference in itsentirety.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Publication No. WO00/08044894, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, herebyincorporated by reference in its entirety.

The oligomeric compounds of the invention may also comprise two or moreof the same, or chemically distinct, sugar, base, and internucleosidelinkage modifications in any combination. The term “chemically distinct”as used herein means different chemical entities whether entirely orpartially distinct. For example, an oligomeric compound may comprise a2′-fluoro and 2′-MOE modification. These two modifications areconsidered to be chemically distinct entities located within the samemolecule.

Modified Nucleobases/Naturally occurring Nucleobases

Oligomeric compounds may also include nucleobase (often referred to inthe art simply as “base” or “heterocyclic base moiety”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesalso referred herein as heterocyclic base moieties include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azguanine and 8-azaadenine, 7-deazaguanine and7-deazaaderine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Suns,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently suitable base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Oligomeric compounds of the present invention can also includepolycyclic heterocyclic compounds in place of one or more heterocyclicbase moieties. A number of tricyclic heterocyclic compounds have beenpreviously reported. These compounds are routinely used in antisenseapplications to increase the binding properties of the modified strandto a target strand. The most studied modifications are targeted toguanosines hence they have been termed G-clamps or cytidine analogs.Many of these polycyclic heterocyclic compounds have the generalformula:

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀=O,R₁₁-R₁₄=H) (Kurchavov, et al., Nucleosides aid Nucleotides, 1997, 16,1837-1846), 1,3-diazaphenothiazine-2-one (R₁₀=S, R₁₁-R₁₄=H), (Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1095, 117,3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀=O,R₁₁-R₁₄=F) (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388). Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance, helicalthermal stability by extended stacking interactions (also see U.S.patent application entitled “Modified Peptide Nucleic Acids” filed May24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled“Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser.No. 10/013,295, both of which are herein incorporated by reference intheir entirety).

Further helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R₁₀=O, R₁₁=−O—(CH₂)₂—NH₂, R₁₂₋₁₄=H)(Lin, K.-Y.; Matteucci, M. J, Am. Chem. Soc. 1998, 120, 8531-8532).Binding studies demonstrated that a single incorporation could enhancethe binding affinity of a model oligonucleotide to its complementarytarget DNA or RNA with a ΔT_(m) of up to 18° relative to 5-methylcytosine (dC5^(mc)), which is the highest known affinity enhancement fora single modification, yet. On the other hand, the gain in helicalstability does not compromise the specificity of the oligonucleotides.The T_(m) data indicate an even greater discrimination between theperfect match and mismatched sequences compared to dC5^(me). It wassuggested that the tethered amino group serves as an additional hydrogenbond donor to interact with the Hoogsteen face, namely the O6, of acomplementary guanine thereby forming 4 hydrogen bonds. This means thatthe increased affinity of G-clamp is mediated by the combination ofextended base stacking and additional specific hydrogen bonding.

Further tricyclic heterocyclic compounds and methods of using them thatare amenable to the present invention are disclosed in U.S. Pat. No.6,028,183 and U.S. Pat. No. 6,007,992, the contents of both areincorporated herein in their entirety.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their uncompromised sequence specificity makes them valuablenucleobase analogs for the development of more potent antisense-baseddrugs. In fact, promising data have been derived from in vitroexperiments demonstrating that heptanucleotides containing phenoxazinesubstitutions are capable to activate RNaseH, enhance cellular uptakeand exhibit an increased antisense activity (Lin, K-Y; Matteucci, M. J.Am. Chem. Soc. 1998, 120, 8531-8532). The activity enhancement was evenmore pronounced in case of O-clamp, as a single substitution was shownto significantly improve the in vitro potency of a 20mer2′-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J. J.;Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc.Natl. Acad. Sci. USA, 1999, 96, 3513-3518). Nevertheless, to optimizeoligonucleotide design and to better understand the impact of theseheterocyclic modifications on the biological activity, it is importantto evaluate their effect on the nuclease stability of the oligomers.

Further modified polycyclic heterocyclic compounds useful asheterocyclic bases are disclosed in but not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692;5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. patentapplication Ser. No. 09/996,292 filed Nov. 28, 2001, certain of whichare herein incorporated by reference.

Conjugates

Oligomeric compounds used in the compositions of the present inventioncan also be modified to have one or more moieties or conjugates forenhancing the activity, cellular distribution or cellular uptake of theresulting oligomeric compounds. In one embodiment such modifiedoligomeric compounds are prepared by covalently attaching conjugategroups to functional groups such as hydroxyl or amino groups. Conjugategroups of the invention include intercalators, reporter molecules,polyamines, polyamides, polyethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugatesgroups include cholesterols, lipids, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes such as including Cy3 and Alexa. Groupsthat enhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve oligomer uptake, enhance oligomerresistance to degradation, and/or strengthen sequence-specifichybridization with RNA. Groups that enhance the pharmacokineticproperties, in the context of this invention, include groups thatimprove oligomer uptake, distribution, metabolism or excretion.Representative conjugate groups are disclosed in International PatentApplication PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure ofwhich is incorporated herein by reference.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. LISA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMVBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al.; Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

The oligomeric compounds of the invention may also be conjugated toactive drug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Oligomeric compounds used in the compositions of the present inventioncan also be modified to have one or more stabilizing groups that aregenerally attached to one or both termini of single-stranded oligomericcompounds or to one or more of the 3′ or 5′ termini of either strand ofa double-stranded compound to enhance properties such as for examplenuclease stability. Included in stabilizing groups are cap structures.By “cap structure or terminal cap moiety” is meant chemicalmodifications, which have been incorporated at either terminus ofoligonucleotides (see for example Wincott et al., WO 97/26270,incorporated by reference herein). These terminal modifications protectthe oligomeric compounds having terminal nucleic acid molecules fromexonuclease degradation, and can help in delivery and/or localizationwithin a cell. The cap can be present at the 5′-terminus (5′-cap) or atthe 3′-terminus (3′-cap) or can be present on both termini. This capstructure is neat to be confused with the inverted methylguanosine“5′cap” present at the 5′ end of native mRNA molecules. In non-limitingexamples, the 5′-cap includes inverted abasic residue (moiety),4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide,4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl riucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety (for more details see Wincott et al.,International PCT publication No. WO 97/26270, incorporated by referenceherein).

3′-cap structures of the present invention include, for example4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide;4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate;1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexylphosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate;1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modifiedbase nucleotide; phosphorodithioate; threo pentofuranosyl nucleotide;acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide;3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety;5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate;1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridgingor non bridging methylphosphonate and 5′-mercapto moieties (for moredetails see Beaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporatedby reference herein).

Further 3′ and 5′-stabilizing groups that can be used to cap one or bothends of an oligomeric compound to impart nuclease stability includethose disclosed in WO 03/004602 published on Jan. 16, 2003.

3′-Endo Modifications

The terms used to describe the conformational geometry of homoduplexnucleic acids are “A Form” for RNA and “B Form” for DNA. The respectiveconformational geometry for RNA and DNA duplexes was determined fromX-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins,Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNAduplexes are more stable and have higher melting temperatures (Tm's)than DNA:DNA duplexes (Sanger et al., Principles of Nucleic AcidStructure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,1997, 25, 2627-2634). The increased stability of RNA has been attributedto several structural features, most notably the improved base stackinginteractions that result from an A-form geometry (Searle et al., NucleicAcids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNAbiases the sugar toward a C3′ endo pucker, i.e., also designated asNorthern pucker, which causes the duplex to favor the A-form geometry.In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and O4′-endo pucker. This isconsistent with Berger, et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a 04′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 49694982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of the duplex formed between a target RNAand a synthetic sequence is central to therapies such as but not limitedto antisense mechanisms including RNAse H, RNAi or any mechanisms thatrequire the binding of a oligomeric compound to an RNA target strand. Inthe case of antisense, effective inhibition of the mRNA requires thatthe antisense compound have a sufficiently high binding affinity withthe mRNA. Otherwise the desired interaction between the oligomericantisense compound and target mRNA strand still occur infrequently,resulting in decreased efficacy.

One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependent on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is furthercorrelated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

In one aspect of the present invention oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA-like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry. There is an apparent preference for an RNA typeduplex (A form helix, predominantly 3′-endo) as a requirement (e.g.trigger) of the RNA interference manchinery which is supported in partby the fact that duplexes composed of 2′-deoxy-2′-F-nucleosides appearefficient in triggering an RNAi response in the C. elegans system.Properties that are enhanced by using more stable 3′-endo nucleosidesinclude, but aren't limited to, modulation of pharmacokinetic propertiesthrough modification of protein binding, protein off-rate, absorptionand clearance; modulation of nuclease stability as well as chemicalstability; modulation of the binding affinity and specificity of theoligomer (affinity and specificity for enzymes as well as forcomplementary sequences); and increasing efficacy of RNA cleavage. Thepresent invention provides oligomeric compounds that can act as triggersof the RNAi pathway having one or more nucleosides modified in such away as to favor a C3′-endo type conformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element (Gallo et al., Tetrahedron (2001), 57, 5707-5713.Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang etal., J. Org. Chem. (1999), 64, 747-754).

Alternatively, preference for the 3′-endo conformation can be achievedby deletion of the 2′-OH as exemplified by 2′deoxy-2′F-nucleosides(Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the3′-endo conformation positioning the electronegative fluorine atom inthe axial position. Other modifications of the ribose ring, for examplesubstitution at the 4′-position to give 4′-F modified nucleosides(Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5,1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or forexample modification to yield methanocarba nucleoside analogs (Jacobsonet al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al.,Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) alsoinduce preference for the 3′-endo conformation. Along similar lines,oligomeric compounds which trigger an RNAi response might be composed ofone or more nucleosides modified in such a way that conformation islocked into a C3′-endo type conformation, i.e. Locked Nucleic Acid (LNA,Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridgedNucleic Acids (ENA™, Morita et a), Bioorganic & Medicinal ChemistryLetters (2002), 12, 73-76.)

The preferred conformation of modified nucleosides and their oligomerscan be estimated by various methods such as molecular dynamicscalculations, nuclear magnetic resonance spectroscopy and CDmeasurements. Hence, modifications predicted to induce RNA-likeconformations, A-form duplex geometry in an oligomeric context, areselected for use in the modified oligonucleotides of the presentinvention. The synthesis of numerous of the modified nucleosidesamenable to the present invention are known in the art (see for example,Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend,1988, Plenum press., and the examples section below.)

In one aspect, the present invention is directed to oligomeric compoundsthat are prepared having enhanced properties, compared to native RNA,against nucleic acid targets. In designing enhanced oligomericcompounds, a target is identified and an oligomeric compound is selectedhaving an effective length and sequence that is complementary to aportion of the target sequence. Each nucleoside of the selected sequenceis scrutinized for possible enhancing modifications. One modificationwould be the replacement of one or more RNA nucleosides with nucleosidesthat have the same 3′-endo conformational geometry but, in addition, anenhancing property. Such modifications can enhance chemical and nucleasestability relative to native RNA while at the same time being muchcheaper and easier to synthesize and/or incorporate into anoligonucleotide. The selected oligomeric compound sequence can befurther divided into regions and the nucleosides of each regionevaluated for enhancing modifications that can be the result of achimeric configuration. Consideration is also given to the 5′ and3′-termini as there are often advantageous modifications that can bemade to one or more of the terminal nucleosides. The oligomericcompounds of the present invention may include at least one 5′-modifiedphosphate group on a single strand or on at least one 5′-positionphosphate of a double-stranded sequence or sequences. Furthermodifications are also considered such as internucleoside linkages,conjugate groups, substitute sugars or bases, substitution of one ormore nucleosides with nucleoside mimetics and any other modificationthat can enhance the affinity of the selected sequence for its intendedtarget.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2-methoxyethoxy(2′-MOE, 2′-OCH₂Cl₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000). One of the immediate advantages of the 2′-MOEsubstitution is the improvement in binding affinity, which is greaterthan many similar 2′ modifications such as O-methyl, O-propyl, andO-aminopropyl. Oligomers having the 2′-O-methoxyethyl substituent alsohave been shown to be antisense inhibitors of gene expression withpromising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995,78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926). Relative to DNA, the oligomers havingthe 2′-MOE modification displayed improved RNA affinity and highernuclease resistance Chimeric oligomers having 2′-MOE substituents in thewing nucleosides and an internal region of deoxy-phosphorothioatenucleotides (also termed a gapped oligomer or gapmer) have showneffective reduction in the growth of tumors in animal models at lowdoses. 2′-MOE substituted oligomers have also shown outstanding promiseas antisense compounds in several disease states. One such MOEsubstituted oligomer is approved for the treatment of CMV retinitis.

Most of the 2′-MOE substituents display a gauche conformation around theC—C bond of the ethyl linker. However, in two cases, a tramsconformation around the C—C bond is observed. The lattice interactionsin the crystal include packing of duplexes against each other via theirminor grooves. Therefore, for some residues, the conformation of the2′-O-substituent is affected by contacts to an adjacent duplex. Ingeneral, variations in the conformation of the substituents (e.g. g⁺ org⁻ around the C—C bonds) create a range of interactions betweensubstituents, both inter-strand, across the minor groove, andintra-strand. At one location, atoms of substituents from two residuesare in van der Waals contact across the minor groove. Similarly, a closecontact occurs between atoms of substituents from two adjacentintra-strand residues.

Previously determined crystal structures of A-DNA duplexes were forthose that incorporated isolated 2′-O-methyl T residues. In the crystalstructure noted above for the 2′-MOE substituents, a conserved hydrationpattern has been observed for the 2′-MOE residues. A single watermolecule is seen located between O2′, O3′ and the methoxy oxygen atom ofthe substituent, forming contacts to all three of between 2.9 and 3.4 Å.In addition, oxygen atoms of substituents are involved in several otherhydrogen bonding contacts. For example, the methoxy oxygen atom of aparticular 2′-O-substituent forms a hydrogen bond to N3 of an adenosinefrom the opposite strand via a bridging water molecule.

In several cases a water molecule is trapped between the oxygen atomsO2′, O3′ and OC′ of modified nucleosides. 2′-MOE substituents with transconformation around the C—C bond of the ethylene glycol linker areassociated with close contacts between OC′ and N2 of a guanosine fromthe opposite strand, and, water-mediated, between OC′ and N3(G). Whencombined with the available thermodynamic data for duplexes containing2′-MOE modified strands, this crystal structure allows for furtherdetailed structure-stability analysis of other modifications.

In extending the crystallographic structure studies, molecular modelingexperiments were performed to study further enhanced binding affinity ofoligonucleotides having 2′-O-modifications. The computer simulationswere conducted on compounds of SEQ ID NO: 10, above, having2′-O-modifications located at each of the nucleosides of theoligonucleotide. The simulations were performed with the oligonucleotidein aqueous solution using the AMBER force field method (Cornell et al.,J. Am. Chem. Soc., 1995, 117, 5179-5197) (modeling software package fromUCSF, San Francisco, Calif.). The calculations were performed on anIndigo2 SOI machine (Silicon Graphics, Mountain View, Calif.).

Another 2′-sugar substituent group that gives a 3′-endo sugarconformational geometry is the 2′-OMe group. 2′-Substitution ofguanosine, cytidine, and uridine dinucleoside phosphates with the 2′-OMegroup showed enhanced stacking effects with respect to the correspondingnative (2′-OH) species leading to the conclusion that the sugar isadopting a C3′-endo conformation. In this case, it is believed that thehydrophobic attractive forces of the methyl group tend to overcome thedestabilizing effects of its steric bulk.

The ability of oligonucleotides to bind to their complementary targetstrands is compared by determining the melting temperature (T_(m)) ofthe hybridization complex of the oligonucleotide and its complementarystrand. The melting temperature (T_(m)) a characteristic physicalproperty of double helices, denotes the temperature (in degreescentigrade) at which 50% helical (hybridized) versus coil (unhybridized)forms are present. T_(m) is measured by using the U spectrum todetermine the formation and breakdown (melting) of the hybridizationcomplex. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption hypochromicity).Consequently, a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the bonds between thestrands.

Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443, havepreviously published a study on the influence of structuralmodifications of oligonucleotides on the stability of their duplexeswith target RNA. In this study, the authors reviewed a series ofoligonucleotides containing more than 200 different modifications thathad been synthesized and assessed for their hybridization affinity andTm. Sugar modifications studied included substitutions on the2′-position of the sugar, 3′-substitution, replacement of the 4′-oxygen,the use of bicyclic sugars, and four member ring replacements. Severalnucleobase modifications were also studied including substitutions atthe 5, or 6 position of thymine, modifications of pyrimidine heterocycleand modifications of the purine heterocycle. Modified internucleosidelinkages were also studied including neutral, phosphorus andnon-phosphorus containing internucleoside linkages.

Increasing the percentage of C3′-endo sugars in a modifiedoligonucleotide targeted to an RNA target strand should preorganize thisstrand for binding to RNA. Of the several sugar modifications that havebeen reported and studied in the literature, the incorporation ofelectronegative substituents such as 2′-fluoro or 2′-alkoxy shift thesugar conformation towards the 3′ endo (northern) pucker conformation.This preorganizes an oligonucleotide that incorporates suchmodifications to have an A-form conformational geometry. This A-formconformation results in increased binding affinity of theoligonucleotide to a target RNA strand.

In addition, for 2′-substituents containing an ethylene glycol motif, agauche interaction between the oxygen atoms around the O—C—C—O torsionof the side chain may have a stabilizing effect on the duplex (Freieribid.). Such gauche interactions have been observed experimentally for anumber of years (Wolfe et al., Acc. Chem. Res., 1972, 5, 102; Abe etal., J. Am. Chem. Soc., 1976, 98, 468). This gauche effect may result ina configuration of the side chain that is favorable for duplexformation. The exact nature of this stabilizing configuration has notyet been explained. While we do not want to be bound by theory, it maybe that holding the O—C—C—O torsion in a single gauche configuration,rather than a more random distribution seen in an alkyl side chain,provides an entropic advantage for duplex formation.

Representative 2′-substituent groups amenable to the present inventionthat give A-form conformational properties (3′-endo) to the resultantduplexes include 2′-O-alkyl, 2′-O-substituted alkyl and 2′-fluorosubstituent groups. Suitable for the substituent groups are variousalkyl and aryl ethers and thioethers, a mines and monoalkyl and dialkylsubstituted amines. It is further intended that multiple modificationscan be made to one or more of the oligomeric compounds of the inventionat multiple sites of one or more monomeric subunits (nucleosides aresuitable) and or internucleoside linkages to enhance properties such asbut not limited to activity in a selected application

Ring structures of the invention for inclusion as a 2′-O modificationinclude cyclohexyl, cyclopentyl and phenyl rings as well as heterocyclicrings having spacial footprints similar to cyclohexyl, cyclopentyl andphenyl rings. 2′-O-substituent groups of the invention included but arenot limited to 2′-O-(trans 2-methoxy cyclohexyl, 2′-O-(trans 2-methoxycyclopentyl, 2′-O-(trans 2-ureido cyclohexyl) and 2′-O-(trans2-methoxyphenyl).

Chemistries Defined

Unless otherwise defined herein, alkyl means C₁-C₁₂, C₁-C₈, or C₁-C₆,straight or (where possible) branched chain aliphatic hydrocarbyl.

Unless otherwise defined herein, heteroalkyl means C₃-C₁₂, C₃-C₈, orC₃-C₆, straight or (where possible) branched chain aliphatic hydrocarbylcontaining at least one, or about 1 to about 3, hetero atoms in thechain, including the terminal portion of the chain. Heteroatoms includeN, O and S.

Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, C₃-C₈, orC₃-C₆, aliphatic hydrocarbyl ring.

Unless otherwise defined herein, alkenyl means C₂-C₁₂, C₂-C₈, or C₂-C₆alkenyl, which may be straight or (where possible) branched hydrocarbylmoiety, which contains at least one carbon-carbon double bond.

Unless otherwise defined herein, alkynyl means C₂-C₁₂, C₂-C₈, orC₂-C₆alkynyl, which may be straight or (where possible) branchedhydrocarbyl moiety, which contains at least one carbon-carbon triplebond.

Unless otherwise defined herein, heterocycloalkyl means a ring moietycontaining at least three ring members, at least one of which is carbon,and of which 1, 2 or three ring members are other than carbon. Thenumber of carbon atoms can vary from 1 to about 12, from 1 to about 6,and the total number of ring members can vary from three to about 15, orfrom about 3 to about 8. Ring heteroatoms are N, O and S.Heterocycloalkyl groups include morpholino, thiomorpholino, piperidinyl,piperazinyl, homopiperidinyl, homopiperazinyl, homomorpholino,homothiomorpholino, pyrrolodinyl, tetrahydrooxazolyl,tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydroisoxazolyl,tetrahydropyrrazolyl, furanyl, pyranyl, and tetrahydroisothiazolyl.

Unless otherwise defined herein, aryl means any hydrocarbon ringstructure containing at least one aryl ring. Aryl rings have about 6 toabout 20 ring carbons. Aryl rings also include phenyl, napthyl,anthracenyl, and phenanthrenyl.

Unless otherwise defined herein, hetaryl means a ring moiety containingat least one fully unsaturated ring, the ring consisting of carbon andnon-carbon atoms. The ring system can contain about 1 to about 4 rings.The number of carbon atoms can vary from 1 to about 12, or from 1 toabout 6, and the total number of ring members can vary from three toabout 15, or from about 3 to about 8. Ring heteroatoms are N, O and S.Hetaryl moieties include pyrazolyl, thiophenyl, pyridyl, imidazolyl,tetrazolyl, pyridyl, pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl,benzimidazolyl, benzothiophenyl, etc.

Unless otherwise defined herein, where a moiety is defined as a compoundmoiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl andalkyl), etc. each of the sub-moieties is as defined herein.

Unless otherwise defined herein, an electron withdrawing group is agroup, such as the cyano or isocyanato group that draws electroniccharge away from the carbon to which it is attached. Other electronwithdrawing groups of note include those whose electronegativitiesexceed that of carbon, for example halogen, nitro, or phenyl substitutedin the ortho- or para-position with one or more cyano, isothiocyanato,nitro or halo groups.

Unless otherwise defined herein, the terms halogen and halo have theirordinary meanings. Halo (halogen) substituents are F, Cl, Br, and I.

The aforementioned optional substituents are, unless otherwise hereindefined, suitable substituents depending upon desired properties.Included are halogens (F, Cl, Br, I), alkyl, alkenyl, and alkynylmoieties, NO₂, NH₃ (substituted and unsubstituted), acid moieties(e.g.—CO₂H, —OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties,aryl moieties, etc.

In all the preceding formulae, the squiggle (˜) indicates a bond to anoxygen or sulfur of the 5′-phosphate. Phosphate protecting groupsinclude those described in U.S. Pat. No. 5,760,209, U.S. Pat. No.5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat.No. 6,326,478, U.S. Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S.Pat. No. 6,465,628 each of which is expressly incorporated herein byreference in its entirety.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides is performedaccording to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA:Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesisas appropriate. In addition specific protocols for the synthesis ofoligomeric compounds of the invention are illustrated in the examplesbelow.

The oligomeric compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The oligomeric compounds of the invention may also be, admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

Salts, Prodrugs and Bioequivalents:

The oligomeric compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive or less active form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes or other chemicals and/or conditions. In particular, prodrugversions of the oligonucleotides of the invention are prepared as SATE((S-acetyl-2-thioethyl) phosphate) derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N-dibetizylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoc acid, naphtalene-2-sulfonicacid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate,glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation ofcyclamates), or with other acid organic compounds, such as ascorbicacid. Pharmaceutically acceptable salts of compounds may also beprepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with a organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine. In one embodiment, double-stranded oligomeric compounds areprovided as sodium salts.

As used herein, the term “patient” refers to a mammal that is afflictedwith one or more disorders associated with survivin expression oroverexpression. It will be understood that the most suitable patient isa human. It is also understood that this invention relates specificallyto the inhibition of mammalian survivin expression or overexpression.

It is recognized that one skilled in the art may affect the disordersassociated with survivin expression or overexpression by treating apatient presently afflicted with the disorders with an effective amountof the compound of the present invention. Thus, the terms “treatment”and “treating” are intended to refer to all processes wherein there maybe a slowing, interrupting, arresting, controlling, or stopping of theprogression of the disorders described herein, but does not necessarilyindicate a total elimination of all symptoms.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” of a compound of the present invention refers to anamount that is effective in treating or preventing the disordersdescribed herein.

The oligomeric compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, a patient, such as a human, suspected of havinga disease or disorder which can be treated by modulating the expressionof survivin is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention cm beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The present invention also includes pharmaceutical compositions andformulations which include oligomeric compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols; including by nebulizer; intratracheal, intranasal,epidermal, intradermal and transdermal, oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection, drip or infusion; orintracranial, e.g., intrathecal or intraventricular, administration.Oligonucleotides with at least one 2′-O-methoxyethyl modification arebelieved to be particularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Compositionsfor oral administration also include pulsatile delivery compositions andbioadhesive composition as described in copending U.S. patentapplication Ser. No. 09/944,493, filed Aug. 22, 2001, and 09/935,316,filed Aug. 22, 2001, the entire disclosures of which are incorporatedherein by reference. Oral administration for treatment of the disordersis described herein. However, oral administration is not the only route.For example, the intravenous route may be desirable as a matter ofconvenience or to avoid potential complications related to oraladministration. When a compound of the present invention is administeredthrough the intravenous route, an intravenous bolus or slow infusion maybe desired.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions and/or formulations comprising theoligomeric compounds of the present invention may also includepenetration enhancers in order to enhance the alimentary delivery of theoligonucleotides. Penetration enhancers may be classified as belongingto one of five broad categories, i.e., fatty acids, bile salts,chelating agents, surfactants and non-surfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, 8, 91-192; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1, 1-33).One or more penetration enhancers from one or more of these broadcategories may be included. Various fatty acids and their derivativeswhich act as penetration enhancers include, for example, oleic acid,lauric acid (C12), capric acid (C10), myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin,caprylic acid, arichidonic acid, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- anddi-glycerides and physiologically acceptable salts thereof (i.e.,oleate, laurate, caprate, myristate, palmitate, stearate, linoleate,etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,1991, 8:2, 91-192; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7:1, 1-33; El-Hariri et al., J. Pharm.Pharmacol., 1992, 44, 651-654). Examples of some fatty acids are sodiumcaprate and sodium laurate, used singly or in combination atconcentrations of 0.5 to 5%.

Various natural bile salts, and their synthetic derivatives, act aspenetration enhancers. Thus, the term “bile salt” includes any of thenaturally occurring components of bile as well as any of their syntheticderivatives. Examples of bile salts are chenodeoxycholic acid (CDCA)and/or ursodeoxycholic acid (UDCA), generally used at concentrations of0.5 to 2%.

Complex formulations comprising one or more penetration enhancers may beused. For example, bile salts may be used in combination with fattyacids to make complex formulations. Suitable combinations include CDCAcombined with sodium caprate or sodium laurate (generally 0.5 to 5%).

Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 92-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; Buur et al., J.Control Rel., 1990, 14, 43-51). Chelating agents have the addedadvantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:2,92-191); and perfluorochemical emulsions, such as FC43 (Takahashi etal., J. Pharm. Pharmacol., 1988, 40, 252-257).

Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl-and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, 8:2, 92-191); andnon-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

A “pharmaceutically acceptable carrier” (excipient) is apharmaceutically acceptable solvent, suspending agent or any otherpharmacologically inert vehicle for delivering one or more nucleic acidsto an animal. The pharmaceutically acceptable carrier may be liquid orsolid and is selected with the planned manner of administration in mindso as to provide for the desired bulk, consistency, etc., when combinedwith a nucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include, butare not limited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodiumstarch glycolate, etc.); or wetting agents (e.g., sodium laurylsulphate, etc.). Sustained release oral delivery systems and/or entericcoatings for orally administered dosage forms are described in U.S. Pat.Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional compatible pharmaceutically-activematerials such as, e.g., antipruritics, astringents, local anestheticsor anti-inflammatory agents, or may contain additional materials usefulin physically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, lichen added, should not unduly interfere with thebiological activities of the components of the compositions of theinvention.

Regardless of the method by which the oligomeric compounds of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of thecompounds and/or to target the compounds to a particular organ, tissueor cell type. Colloidal dispersion systems include, but are not limitedto, macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes and lipid:oligonucleotide complexes ofuncharacterized structure. One colloidal dispersion system is aplurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layer(s) made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech., 1995, 6, 698-708).

Certain embodiments of the invention provide for liposomes and othercompositions one or more other chemotherapeutic agents which function bya non-antisense mechanism. Examples of such chemotherapeutic agentsinclude but are not limited to daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclopiosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin, carboplatin anddiethylstilbestrol (DES). Sec, generally, The Merck Manual of Diagnosisand Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway,N.J. When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention. See,generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkowet al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49,respectively). Other non-antisense chemotherapeutic agents are alsowithin the scope of this invention. Two or more combined compounds maybe used together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art,Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly. Persons of ordinary skill in the art can easilyestimate repetition rates for dosing based on measured residence timesand concentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 μg to 100 g per kg of body weight, once or more daily,weekly, monthly, or yearly. For double-stranded compounds, the dose mustbe calculated to account for the increased nucleic acid load of thesecond strand (as with compounds comprising two separate strands) or theadditional nucleic acid length (as with self complementary singlestrands having double-stranded regions).

Double-stranded compounds can be introduced into cells in a number ofdifferent ways. For example, the double-stranded compounds can beadministered by microinjection; bombardment by microparticles covered bythe double-stranded compounds; soaking the cells in a solution of thedouble-stranded compounds; electroporation of cells in the presence ofthe double-stranded compounds; liposome-mediated delivery ofdouble-stranded compounds; transfection mediated by chemicals such ascalcium phosphate, cationic lipids, etc.; viral infection;transformation; and the like. The double-stranded compounds can beintroduced along with components that enhance RNA uptake by the cells,stabilize the annealed strands, or otherwise increase the inhibition offunction of the target polynucleotide sequence. In the case of a cellculture or tissue expoant, the cells are conveniently incubated in asolution containing the double-stranded compounds, or subjected tolipid-mediated transformation.

Determination of the optimal amounts of double-stranded compounds to beadministered to human or animal patients for the prevention or treatmentof pathologies associated with survivin expression or overexpression, aswell as methods of administering therapeutic or pharmaceuticalcompositions comprising such double-stranded oligonucleotides, is withinthe skill of those in the pharmaceutical art, Dosing of a human oranimal patient is dependent on the nature of the symptom, condition, ordisease; the nature of the infected cell or tissue; the patient'scondition; body weight; general health; sex; diet; time, duration, androute of administration; rates of absorption, distribution, metabolism,and excretion of the double-stranded compounds; combination with otherdrugs; severity of the pathology; and the responsiveness of the diseasestate being treated. The amount of double-stranded compoundsadministered also depends on the nature of the target polynucleotidesequence or region thereof, and the nature of the double-strandedcompounds, and can readily be optimized to obtain the desired level ofeffectiveness. The course of treatment can last from several days toseveral weeks or several months, or until a cure is effected or anacceptable diminution or prevention of the disease state is achieved.Optimal dosing schedules can be calculated from measurements of drugaccumulation in the body of the patient in conjunction with theeffectiveness of the treatment. Persons of ordinary skill can easilydetermine optimum dosages, dosing methodologies, and repetition rates.

While the embodiments of the invention have been described withspecificity in accordance with certain of the embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-Alkoxy Amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham, Mass. orGlen Research, Inc. Sterling, Va.). Other 2′-O-alkoxy substitutednucleoside amidites are prepared as described in U.S. Pat. No.5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods (Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham, Mass.).

2′-Fluoro Amidites 2′-Fluorodeoxytidenosine Amidites

2′-fluoro oligonucleotides were synthesized as described previously(Kawasaki, et. al, J. Med. Chem., 1993, 36, 831-841) and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom was introduced by a S_(N)2 displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyrylarabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine.

Selective O-deacylation and triflation was followed by treatment of thecrude product with fluoride, then deprotection of the THP groups.Standard methodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) Modified Amidites

2′-O-Methoxyethyl-substituted nucleoside amidites were prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro(1-(beta-D-arabinofuranosyl)-5-methyluridine)

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under-reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a White solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 a 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/Acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/Acetone (5:5:1) containing 0.5% Et₃NM. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by the by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside.

Triethylamine (189 mL, 1.44 M) wag added to a solution of triazole (90g, 1.3 M) in CH₃CN (1 L), cooled to −5° C. and stirred for 0.5 hoursusing an overhead stirrer. POCl₃ was added dropwise, over a 30 minuteperiod, to the stirred solution maintained at 0-10° C., and theresulting mixture stirred for an additional 2 hours. The first solutionwas added dropwise, over a 45 minute period, to the latter solution. Theresulting reaction mixture was stored overnight in a cold room. Saltswere filtered from the reaction mixture and the solution was evaporated.The residue was dissolved in EtOAc (1 L) and the insoluble solids wereremoved by filtration. The filtrate was washed with 1×300 mL of NaHCO₃and 2×300 mL of saturated NaCl, dried over sodium sulfate andevaporated. The residue was triturated with EtOAc to give the titlecompound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyleytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g,0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra(iso-propyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (tic showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-(Aminooxyethyl) nucleoside amidites and 2′-(dimethylaminoooxyethyl)nucleoside amidites

Aminooxyethyl and dimethylaminooxyethyl amidites are prepared as per themethods of U.S. Pat. No. 6,127,533 which is herein incorporated byreference.

Example 2 Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides weresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphor-amidite chemistry with oxidation byiodine.

Phosphorothioates (P═S) were synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 hours), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described itpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference. 3′-Deoxy-3′-amino phosphoramidate oligonucleotides areprepared as described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

4-Ribonucleoside and 2′-deoxy-4′-ribonucleoside compositions may be madeby the method taught by Naka et al., J. Am. Chem. Soc. 122:7233-7243,2000 and U.S. Pat. No. 5,639,873, which are incorporated by referenceherein in their entirety.

Example 3 Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference. The oligomeric compounds of theinvention may also comprise mixed linkages in which any number of two ormore types of linkages are present in any order and at any positionwithin the oligomeric compound, for example the 5′ half of the compoundcomprising phosphorothioate linkages and the 3′ half comprisingphosphodiester linkages. These are referred to as mixed phosphorothioateand phosphodiester linkages

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262: herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

Double-stranded compounds of the invention can be of several typesincluding but not limited to, siRNAs, canonical siRNAs, blunt-endedsiRNAs or hairpins. Single-stranded compounds of the invention whichelicit the RNAi antisense mechanism are also within the scope of theinvention. These include, but are not limited to, ssRNAi and antisenseRNA (asRNA).

(2′-O-Me)-(2′-deoxy)-(2′-O-Me)chimeric phosphorothioate oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for the 2′-MOEmodified nucleotides. The standard synthesis cycle is modified byincreasing the wait step after the delivery of tetrazole and base to 600s repeated four times for RNA and twice for 2′-O-methyl. The fullyprotected oligonucleotide is cleaved from the support and the phosphategroup is deprotected in 3:1 Ammonia/Ethanol at room temperatureovernight then lyophilized to dryness. Treatment in methanolic ammoniafor 24 hours at room temperature is then done to deprotect all bases andsample was again lyophilized to dryness. The pellet is resuspended in IMTBAF in THF for 24 hours at room temperature to deprotect the 2′positions. The reaction is then quenched with 1M TEAA and the sample isthen reduced to ½ volume by rotovac before being desalted on a G25 sizeexclusion column. The oligo recovered is then analyzedspectrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

(2′-O-(2-Methoxyethyl))-(2′-deoxy)(2′-O-(Methoxyethyl))chimericphosphorothioate oligonucleotides

(2′-O-(2-methoxyethyl))-(2′-deoxy)-(−2′-O-(methoxyethyl))chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

(2′-O-(2-Methoxyethyl)Phosphodiester)-(2′-deoxyPhosphorothioate)(2′-O-(2′-Methoxyethyl)Phosphodiester) chimericoligonucleotides

(2′-O-(2-methoxyethyl phosphodiester)-(2′-deoxyphosphorothioate)-(2′-O-(methoxyethyl)phosphodiester) chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference,

RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized n a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is wasted and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups-on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%nethylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chest. Soc., 1998, 120, 11820-11821; Matteucei, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedron Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides, whether single or doublestranded) of the present invention can be synthesized by the methodsherein or purchased from Dharmacon Research, Inc (Lafayette, Colo.).Once synthesized, complementary RNA antisense compounds can then beannealed by methods known in the art to form double-stranded (duplexed)antisense compounds. For example, duplexes can be formed by combining 30μl of each of the complementary strands of RNA oligonucleotides (50 uMRNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mMpotassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate)followed by heating for 1 minute at 90° C., then 1 hour at 37° C. Theresulting duplexed antisense compounds can be used in kits, assays,screens, or other methods to investigate the role of a target nucleicacid, or for diagnostic or therapeutic purposes.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis 96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1 μl dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis 96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACEJ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACEJ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used.

MCF7:

The human breast carcinoma cell line MCF-7 was obtained from theAmerican Type Culture Collection (Manassas, Va.). MCF-7 cells wereroutinely cultured in DMEM low glucose (Invitrogen Life Technologies,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenLife Technologies, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached about 90% confluence.Cells were seeded into 96-well plates (Falcon-Primaria #3872) at adensity of about 7000 cells/well for use in RT-PCR analysis. ForNorthern blotting or other analyses, cells may be seeded onto 100 mm orother standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

HeLa Cells:

The human epitheloid carcinoma cell line HeLa was obtained from theAmerican Tissue Type Culture Collection (Manassas, Va.). HeLa cells wereroutinely cultured in DMEM, high glucose (Invitrogen Corporation.,Carlsbad, Calif.) supplemented with 10% fetal bovine serum (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 90%confluence. Cells were seeded into 24-well plates (Falcon-Primaria#3846) at a density of approximately 50,000 cells/well or in 96-wellplates at a density of approximately 5,000 cells/well for use in RT-PCRanalysis. For Northern blotting or other analyses, cells were harvestedwhen they reached approximately 90% confluence.

U-87 MG Cells:

The human glioblastoma U-87 MG cell line was obtained from the AmericanType Culture Collection (Manassas, Va.). U-87 MO cells were cultured inDMEM (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.)and antibiotics. Cells were routinely passaged by trypsinization anddilution when they reached appropriate confluence. Cells were seededinto 96-well plates (Falcon-Primaria #3872) at a density of about 10,000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide. For Northern blottingor other analyses, cells may be seeded onto 100 mm or other standardtissue culture plates and treated similarly, using appropriate volumesof medium and oligonucleotide.

HUVEC Cells:

HUVEC were obtained from ATCC and routinely cultured in EBM (CloneticsCorp, Walkersville, Md.) supplemented with SingleQuots supplements.Cells were routinely passaged by trypsinization and dilution when theyreached 90% confluence were maintained for up to 15 passages. For cellsgrown in 96-well plates (10,000 cells/well), wells were washed once with200 μL OPTI-MEM-1™ reduced-serum medium (Gibco BRL) and then treatedwith 130 μL of OPTI-MEM-1™ containing 12 μg/mL LIPOFECTIN™ (Gibco BRL)and the desired double-stranded compounds at a final concentration of 25nM. After 5 hours of treatment, the medium was replaced with freshmedium. Cells were harvested 16 hours after dsRNA treatment, at whichtime RNA was isolated and target reduction measured by RT-PCR.

Treatment with Oligomeric Compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEMJ-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM-1 containing 3.75 μg/mL LIPOFECTINJ(Gibco BRL) and the desired oligonucleotide at a final concentration of150 nM. For dsRNA compounds, 2×130 μL of OPTI-MEM-1 was used. After, 4hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16 hours after oligonucleotide treatment. Theconcentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control RNAse H oligonucleotide is ISIS 13920,TCCGTCATCGCTCCTCAGGC, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer(2-O-methoxyethyls shown in bold) with a phosphorothioate backbone whichis targeted to human H-ras. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition or H-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNAis then utilized as the screening concentration for new oligonucleotidesin subsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration hi subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10 Analysis of Oligonucleotide Inhibition of Survivin Expression

Antisense modulation of survivin expression can be assayed in a varietyof ways known in the art. For example, survivin mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.14.2.9 and 4.5.14.5.3, John Wiley & Sons,Inc., 1993. Northern blot analysis is routine in the art and is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions. Other methods of PCR are alsoknown in the art.

Survivin protein levels can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).Antibodies directed to survivin can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of Monoclonal Antibodies is Taughtin, for Example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found a,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11 Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% IP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μl of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wasp buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

Total mRNA was isolated using an RNEASY 96 kit and buffers purchasedfrom Qiagen, Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pippeting three times up anddown. The samples were then transferred to the RNEASY 96 well plateattached to a QIAVAC manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96 plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96 plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC manifold and blotted dry on paper towels. The plate was thenreattached to the QIAVAC manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 mL water.

Example 13 Real-Time Quantitative PCR Analysis of Survivin mRNA Levels

Quantitation of survivin mRNA levels was determined by real-timequantitative PCR using the ABI PRISM 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE or FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the Si end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular (six-second) intervals bylaser optics built into the ABI PRISM 7700 Sequence Detection System. Ineach assay, a series of parallel reactions containing serial dilutionsof mRNA from untreated control samples generates a standard curve thatis used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1×TAQMAN buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension). Probes and primers to human survivinwere designed to hybridize to a human survivin sequence, using publishedsequence information (GenBank accession number U75285, incorporatedherein as SEQ ID NO:3). For human survivin the PCR primers were: forwardprimer: AAGGACCACCGCATCTCTACA (SEQ ID NO: 4) reverse primer:CCAAGTCTGGCTCGTTCTCAGT (SEQ ID NO: 5) and the PCR probe was:FAM-CGAGGCTGGCTTCATCCACTGCC-TAMRA (SEQ ID NO: 6) where FAM (PB-AppliedBiosystems, Foster City, Calif.) is the fluorescent reporter dye) andTAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.For human GAPDH the PCR primers were: forward primer:GAAGGTGAAGGTCGCIAGTC (SEQ ID NO: 7) reverse primer: GAAGATGGTGATGGGATFTC(SEQ ID NO: 8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA3′ (SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.)is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems,Foster City, Calif.) is the quencher dye.

Example 14 Western Blot Analysis of Survivin Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 hours after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to survivin is used,with a radiolabelled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale, Calif.).

Example 15 Design and Screening of Double-Stranded Antisense Compounds(siRNAs) Targeting Survivin

In accordance with the present invention, a series of double-strandedoligomeric compounds (siRNAs) comprising the antisense compounds of thepresent invention and their complements can be designed to targetsurvivin. The nucleobase sequence of the antisense strand of the duplexcomprises at least a portion of an oligonucleotide targeted to survivinas described herein. The ends of the strands may be modified by theaddition of one or more natural or modified nucleobases to form anoverhang. The sense strand of the dsRNA is then designed and synthesizedas the complement of the antisense strand and may also containmodifications or additions to either terminus. For example, in oneembodiment, both strands of the dsRNA duplex would be complementaryoffer the central nucleobases, each having overhangs at one or bothtermini.

For example, a duplex comprising an antisense strand having thesequence: CGAGAGGCGGACGGGACCG (SEQ ID NO: 10) and having atwo-nucleobase overhang of deoxythymidine(dT) would have the followingstructure:

cgagaggcggacgggaccgTT Antisense (SEQ ID NO:11) TTgctctccgcctgccctggcComplement (SEQ ID NO:12)As shown, this double-stranded compound represents a canonical siRNA.

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 10) may be preparedwith blunt ends (no single-stranded overhang) as shown:

cgagaggcggacgggaccg Antisense (SEQ ID NO:10) gctctccgcctgccctggcComplement (SEQ ID NO:13)As shown, this double-stranded compound represents a blunt-ended siRNA.

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds (siRNAs) are evaluatedfor their ability to modulate survivin expression according to theprotocols described herein.

Cell Culture Conditions, Determination of IC₅₀ Values for dsRNAs InVitro

IC50

In vitro IC₅₀ values for dsRNAs of the present invention can bedetermined by contacting in vitro varying concentrations of dsRNAs andappropriate cell lines, tissues, or organs exhibiting pathologiesassociated with survivin expression or overexpression, and determiningthe quantitative effect(s) of these dsRNAs at such concentrations onparameters including, but not limited to, various steps, stages, oraspects of survivin pathology or pathogenesis. Representative parametersthat can be studied include, for example, translation of survivin mRNAsor survivin protein synthesis; effect on surrogate markers; or any otherparameter that is indicative of potential dsRNA therapeuticeffectiveness that can be conveniently measured in vitro.

Given the state of the art, it should be possible for one of ordinaryskill to either adapt currently existing cell-based assays, or developcompletely novel in vitro assays, to determine IC₅₀ values for thedsRNAs disclosed herein without undue experimentation.

Western Blots:

For western blot analysis, cells are plated in 10 cm tissue culturedishes Falcon, #3003) at a density of 7.5×10⁵ cells/dish. For RT-PCRanalysis, 96-well plates (Corning Incorporated, #3596) are plated with1×10⁴ cells/well. Lipofactin (GIBCO/Invitrogen) transfection reagent isused at a concentration of 3 ul/ml OPTIMEM reduced serum medium(Gibco/Invitrogen)/100 nM siRNA duplex. Lipofectin reagent is incubatedwith OPTIMEM medium for 30 minutes prior to the addition of siRNA. Thedesired amount of siRNA is added and mixed. Further 1:1 dilutions areperformed in OPTIMEM. Cells are washed twice with 1× phosphate bufferedsaline and then treated with the siRNA/Lipofectin mixture in OPTIMEM.After a 4 hr incubation period, OPTIMEM medium is replaced with completegrowth medium. Cells are harvested after additional 16-20 hrs forWestern blot or RT-PCR analysis.

At the end of the incubation period culture medium is removed and cellsare washed twice with PBS. Cells are lysed in RIPA buffer (20 mMTris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 10 mM Sodium Pyrophosphate, 50mM Sodium Fluoride, and 1% Nonidet P40) plus Complete™ Mini proteaseinhibitor tablets (Roche, #186153) and 1 mM Sodium orthovanadate (Sigma)by directly adding buffer to the plate. Lysates are collected byscrapping, transferred to microfuge tubes and cleared from the cellulardebris by centrifugation at 14,000 rpm for 30 minutes at 4° C. Proteinconcentrations are determined using BCA protein assay reagents (Pierce).Total cellular protein is subjected to SDS-PAGE and transferred ontoImmobilon-P membranes (Millipore, #IPVH091). Membranes are probed withprimary antibodies to survivin (R&D Systems, #AF886) and to β-Actin(Sigma, #A5441). Horseradish peroxidase linked Anti-rabbit Ig, andanti-mouse Ig, antibodies (Pharmacia) are used as secondary antibodies.Antigen-antibody complexes are visualized by incubation of membrane inSuperSignal West Pico chemiluminescence reagents (Pierce) for 5 minutesfollowed by capturing of the chemiluminescence using Fluor-S imager(Biorad) equipped with the cool CCD camera. The protein bands ofinterest for each sample are quantified using Quantity One software(Biorad). The percentage inhibition for each samples are calculated bycomparing the survivin/Actin protein ratio for the sample tosurvivin/Actin protein ratio for the untreated control. The IC₅₀ isderived from the non-linear regression analysis of the percentageinhibition data using GraphPad Prism software (GraphPad Software).

RNA Isolation:

Total RNA is isolated using RNeasy® 96 kit according to recommendedprotocol (Qiagen). Briefly, cells are washed with 200 ul PBS afterremoval of growth medium. Following washing, 100 μl buffer RLT is added,plate is shaken vigorously for about 20 seconds. To each well 100 μl 70%ethanol are added and mixed by pipetting up and down three times.Samples are then applied into the wells of the RNeasy 96 plate placed inthe QIAvac top base of the QIAvac 96 manifold, which is attached to avacuum source. Vacuum is applied for 30 seconds or until transfer iscomplete. To each well 80 μl of DNase I incubation mix (20 mM Tris-HCl,pH 8.4, 2 mM MgCl2, 50 mM KCl and 225 Units/ml DNase I from Invitrogen)is added and incubated at room temperature for 30 minutes. Buffer RW1 (1ml/well) is added and the vacuum is applied for 30 seconds. Buffer RPE(1 ml/well) is added to each well and vacuum is applied for 30 seconds.Washing steps with buffer RW1 and RPE1 are repeated once more. The plateis then removed from the manifold and blotted dry on paper towels. Theplate is placed back in the QIAvac manifold and the vacuum is appliedfor 10 minutes. To elute RNA 30 μl of RNase-free water is added directlyonto the membrane of each well, incubated for 1 minutes and vacuum isapplied for 30 seconds. In order to maximize the recovery of total RNAthe elution step is repeated with additional 30 μl/well RNase-freewater.

Quantitation of Surviving:

Quantitation of survivin and GAPDH mRNA levels is determined byreal-time quantitative RT-PCR using the ABI PRISM® 7900 SequenceDetection System (Applied Biosystems). RT-PCR reactions are carried outby adding 15 ul of TaqMan One Step PCR Master Mix (Applied Biosystems,#4309169) reagents (containing 100 nM each of forward primer and reverseprimer, and 200 nM probe) to 96-well plates with 10 μl total RNA. Forhuman survivin, the forward PCR primer is: 5′GCACCACTTCCAGGGATTATrC3′(SEQ ID NO: 186), and the reverse primer is:5′TCTCCTTTCCTAAGACATTGCTAAG3′ (SEQ ID NO: 187). The survivin TaqMan®probe used is 5′(FAM)TGGTGCCACCAGCCTTCCTGTG3′ (SEQ ID NO: 188)(Biosearch Technologies, Inc.). This primer-probe set, designed to SEQID No 14 was used for all experiments in Examples 15 to the end. Forhuman GAPDH, TaqMan GAPDH Control Reagent Kit is used (AppliedBiosystems, #402869). The percentage inhibition for each samples arecalculated by comparing the survivin/GAPDH mRNA ratio for the sample tosurvivin/GAPDH mRNA ratio for the untreated control. The IC₅₀ is derivedfrom the non-linear regression analysis of the percentage inhibitiondata using GraphPad Prism software (GraphPad Software).

Example 16 Design of Phenotypic Assays and In Vivo Studies for the Useof Survivin Inhibitors Phenotypic Assays

Once active oligomeric compounds targeting survivin have been identifiedby the methods disclosed herein, the compounds are further investigatedin one or more phenotypic assays, each having measurable endpointspredictive of efficacy in the treatment of a particular disease state orcondition.

Phenotypic assays, kits and reagents further use are well known to thoseskilled in the art and are herein used to investigate the role and/orassociation of survivin in health and disease. Representative phenotypicassays, which can be purchased from any one of several commercialvendors, include those for determining cell viability, cytotoxicity,proliferation or cell survival (Molecular Probes, Eugene, Oreg.;PerkinElmer, Boston, Mass.), protein-based assays including enzymaticassays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes,N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation,signal transduction, inflammation, oxidative processes and apoptosis(Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation(Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formationassays, cytokine and hormone assays and metabolic assays (ChemiconInternational Inc., Temecula, Calif.; Amersham Biosciences, Piscataway,N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withsurvivin inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the geneotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the survivin inhibitors.Hallmark genes, or those genes suspected to be associated with aspecific disease state, condition, or phenotype, are measured in bothtreated and untreated cells.

Example 17 Modulation of Human Survivin Expression by Double-StrandedRNA (dsRNA)

In accordance with the present invention, a series of double-strandedoligomeric compounds comprising the antisense compounds of the presentinvention and their complements thereof, were designed to targetsurvivin mRNA. The sense strand of the dsRNA is designed and synthesizedas the reverse complement of the antisense strand, a list of which isshown in Table 1. The oligomeric compounds were evaluated in HeLa cells.Culture methods used for HeLa cells are found, for example, atwww.atcc.org.

For cells grown in 96-well plates, wells were washed once with 200 μLOPTI-MEM-1™ reduced-serum medium (Gibco BRL) and then treated with 130μL of OPTI-MEM-1™ containing 12 μg/mL LIPOFECTIN™ (Gibco BRL) and thedesired dsRNA at a final concentration of 25 nM. After 5 hours oftreatment, the medium was replaced with fresh medium. Cells wereharvested 16 hours after dsRNA treatment, at which time RNA was isolatedand target reduction measured by RT-PCR as described above.

The antisense sequences of the dsRNA oligomeric compounds are shown inTable 1. Prior to treatment of the HeLa cells, the dsRNA oligomers weregenerated by annealing the antisense and sense strands according to themethod outlined in Example 16. Target sites are indicated by the first(5′ most) nucleotide number, as given in the sequence source reference(Genbank accession no. NM_(—)001168.1, incorporated herein as SEQ ID NO:14), to which the antisense strand of the dsRNA oligonucleotide binds.

All compounds in Table 1 are dsRNAs, 20 nucleotides in length with theantisense strand listed first (top strand) in the 5′ to 3′ orientation,and the sense strand lifted second (bottom strand), also in the 5′ to 3′orientation. All nucleosides are ribose and backbone linkages arephosphate (P═O). “Target site” refers to the 5′-most position of thetarget region on the survivin mRNA to which the antisense strand istargeted. As such, these compounds are blunt-ended siRNAs.

Data were obtained by real-time quantitative PCR as described herein.HeLa cells were treated with double stranded oligomeric compounds(composed of antisense strands hybridized to their corresponding sensestrands) targeting human survivin mRNA.

TABLE 1 Inhibition of human survivin mRNA levels by dsRNA oligomericcompounds TARGET SEQ ID % Strand ISIS # REGION SITE SEQUENCE NO INHIB AS339044 coding 67 agggcugccaggcagggggc 15 ND S 339074 gccccugccuggcagcccu16 AS 339045 coding 456 auccauggcagccagcugcu 17 86 S 339075agcagcuggcugccauggau 18 AS 339046 3′UTR 512 aacccuggaaguggugcagc 19 28 S339076 gcugcaccacuuccaggguu 20 AS 339047 3′UTR 534 aggcugguggcaccagggaa21  9 S 339077 uucccuggugccaccagccu 22 AS 339048 3′UTR 586auuugaaaauguugaucucc 23 86 S 339078 ggagaucaacauuuucaaau 24 AS 3390493′UTR 605 agcacaguugaaacaucuaa 25 71 S 339079 uuagauguuucaacugugcu 26 AS339050 3′UTR 642 agaagcaccucuggugccac 27 51 S 339080guggcaccagagguqcuucu 28 AS 339051 3′UTR 756 ucccucacuucucaccuggu 29 48 S339081 accaggugagaagugaggga 30 AS 339052 3′UTR 780 gcaaaagggacacugccuuc31 85 S 339082 gaaggcagugucccuuuugc 32 AS 339053 3′UTR 815aggcucugcccacgcgaaca 33 21 S 339083 uguucgcgugggcagagccu 34 AS 3390543′UTR 846 caacaugagguccagacaca 35 50 S 339084 ugugucuggaccucauguug 36 AS339055 3′UTR 876 aguccacacucaggacugug 37 59 S 339085cacaguccugaguguggacu 38 AS 339056 3′UTR 914 uaaggaaccugcagcucaga 39 86 S339086 ucugagcugcagguuccuua 40 AS 339057 3′UTR 1030 cagggacucugucuccauuc41 48 S 339087 gaauggagacagagucccug 42 AS 339058 3′UTR 1070aacaaaauaagaaagccaug 43 78 S 339088 cauggcuuucuuauuuuguu 44 AS 3390593′UTR 978 gcuauucugugaauuaacaa 45 65 S 339089 uuguuaauucacagaauagc 46 AS339060 3′UTR 1101 aguuugugcuauucugugaa 47 85 S 339090uucacagaauagcacaaacu 48 AS 339061 3′UTR 1130 agaauggcuuugugcuuagu 49 48S 339091 acuaagcacaaagccauucu 50 AS 339062 3′UTR 1165uccaccugaaguucaccccg 51 73 S 339092 cggggugaacuucaggugga 52 AS 3390633′UTR 1212 aaggaguaucugccagacgc 53 67 S 339093 gcgucuggcagauacuccuu 54AS 339064 3′UTR 1232 uaaucacacagcaguggcaa 55 73 S 339094uugccacugcugugugauua 56 AS 339065 3′UTR 1258 gugccccgcggcucacuggg 57 19S 339095 cccagugagccgcggggcac 58 AS 339066 3′UTR 1305aaaggauuuaggccacugcc 59 73 S 339096 ggcaguggccuaaauccuuu 60 AS 3390673′UTR 1329 acagcaucgagccaagucau 61 63 S 339097 augacuuggcucgaugcugu 62AS 339068 3′UTR 1366 acagacacacggccugcagc 63 20 S 339098gcugcaggccgugugucugu 64 AS 339069 3′UTR 1394 gaacgugacagaugugaagg 65 43S 339099 ccuucacaucugucacguuc 66 AS 339070 3′UTR 1487uccaucaucuuacgccagac 67 74 S 339100 gucuggcguaagaugaugga 68 AS 3390713′UTR 1498 ggcgaaucaaauccaucauc 69 57 S 339101 gaugauggauuugauucgcc 70AS 339072 3′UTR 1529 auccacccugcagcucuaug 71 31 S 339102cauagagcugcaggguggau 72 AS 339073 3′UTR 1569 gccgagaugaccuccagagg 73 50S 339103 ccucuggaggucaucucggc 74 AS 341881 3′UTR 584uuugaaaauguugaucucctt 75 93 S 341880 ggagaucaacauuuucaaatt 76 AS 3418833′UTR 1068 acaaaauaagaaagccaugtt 77 80 S 341882 cauggcuuucuuauuuugutt 78AS 341885 3′UTR 1303 aaggauuuaggccacugcctt 79 78 S 341884ggcaguggccuaaauccuutt 80

All dsRNA compounds except ISIS 339046, 339047, 339053, 339065, 339068and 339072 demonstrated over 45% inhibition of survivin expression.

In a dose-response experiment, HeLa cells were treated with 1.1, 3.3, 10and 30 nM of the indicated oligonucleotide mixed with 3 ug/mL LIPOFECTINper 100 nM oligonucleotide as described by other examples herein.Untreated cells served as controls. Following 16 hours of treatment, RNAwas prepared from cells for subsequent real-time PCR analysis.

Human survivin mRNA expression levels were quantitated by real-time PCRand gene target quantities were normalized using Ribogreen as describedin other examples herein. Data are averages from two experiments and areshown in Table 2.

The identity of the two strands of the duplex are shown separated by anunderscore, with the antisense strand shown first (antisensestrand_sense strand).

TABLE 2 Inhibition of human survivin mRNA levels by dsRNA oligomericcompounds: dose response Percentage of Inhibition of survivin expressionin HeLa cells Oligonucleotide Concentration 1.1 3.3 10 30 Isis # nM nMnM nM 339045_339075 24 44 62 81 339048_339078 42 59 74 83 339052_33908226 55 70 83 339056_339086 35 54 72 81 339058_339088 22 44 63 75339060_339090 20 50 70 85

As shown in Table 2, the compounds tested inhibit human survivin mRNAexpression in HeLa cells in a concentration-dependent manner.

Example 18 Modulation of Human Survivin Expression by Double-StrandedRNA (dsRNA) with a 5′-phosphate Cap

In accordance with the present invention, a series of double-strandedoligomeric compounds comprising the antisense compounds shown in Table 1(ISIS 339045-339073), each modified with a 5′-terminal phosphate group,and the complements thereof, were designed to target survivin mRNA. Thecorresponding dsRNA compounds are ISIS 341201-341229 (Table 3). Thesense strand of the dsRNA was designed and synthesized as the complementof the antisense strand, a list of which is shown in Table 2. Theoligomeric compounds were evaluated in HeLa cells. Culture methods usedfor HeLa cells are found, for example, at www.atcc.org.

For cells grown in 96-well plates, wells were washed once with 200 uLOPTI-MEM-1™ reduced-serum medium (Gibco BRL) and then treated with 130μL of OPTI-MEM-1™ containing 12 μg/mL LIPOFECTIN™ (Gibco BRL) and thedesired dsRNA at a final concentration of 25 nM. After S hours oftreatment, the medium was replaced with fresh medium. Cells wereharvested 16 hours alter dsRNA treatment, at which time RNA was isolatedand target reduction measured by RT-PCR.

The dsRNA oligomeric compounds are shown in Table 3. Prior to treatmentof the HeLa cells, the dsRNA oligomers were generated by annealing theantisense and sense strands according to the method outlined in Example17. Target sites are indicated by the first (5′ most) nucleotide number,as given in the sequence source reference (Genbank accession no.NM_(—)001168.1, incorporated herein as SEQ ID NO: 14), to which theantisense strand of the dsRNA oligonucleotide binds.

All compounds in Table 3 are oligoribonucleotides, 20 nucleotides inlength with the antisense strand shown first, and the sense strand shownsecond, both in the 5′ to 3′ direction. Compounds in Table 3 havephosphate (P═O) backbones and also comprise a terminal 5′-phosphate capon each strand. The compounds in Table 3 are blunt-ended siRNAs.

Data were obtained by real-time quantitative PCR as described in otherexamples herein. HeLa cells were treated with double stranded oligomericcompounds targeting human survivin mRNA.

TABLE 3 Inhibition of human survivin mRNA levels by dsRNA oligomericcompounds with a 5′-phosphate cap SEQ ID % Strand ISIS # REGION SEQUENCENO INHIB AS 341201 Coding auccauggcagccagcugcu 17 85 S 341231agcagcuggcugccauggau 18 AS 341202 3′UTR aacccuaggaguggugcagc 19 42 S341232 gcugcaccacuuccaggguu 20 AS 341203 3′UTR aggcugguggcaccagggaa 2123 S 341233 uucccuggugccaccagccu 22 AS 341204 3′UTR auuugaaaauguugaucucc23 84 S 341234 ggagaucaacauuuucaaau 24 AS 341205 3′UTRagcacaguugaaacaucuaa 25 78 S 341235 uuagauuuuucaacugugcu 26 AS 3412063′UTR agaagcaccucuggugccac 27 61 S 341236 guggcaccagaggugcuucu 28 AS341207 3′UTR ucccucacuucucaccuggu 29 58 S 341237 accaggugagaagugaggga 30AS 341208 3′UTR gcaaaagggacacugccuuc 31 84 S 341238 gaaggcagugucccuuuugc32 AS 341209 3′UTR aggcucugcccacgcgaaca 33 33 S 341239uguucgcgugagcagagccu 34 AS 341210 3′UTR caacaugagguccagacaca 35 66 S341240 ugugucuggaccucauguug 36 AS 341211 3′UTR aguccacacucaggacugug 3771 S 341241 cacaguccugaguguggacu 39 AS 341212 3′UTR uaaggaaccugcagcucaga39 85 S 341242 ucugagcugcagguuccuua 40 AS 341213 3′UTRcagggacucugucuccauuc 41 52 S 341243 gaauggagacagagucccug 42 AS 3412143′UTR aacaaaauaagaaagccaug 43 85 S 341244 caugccuuucuuauuuuguu 44 AS341215 3′UTR gcuauucugugaauuaacaa 45 71 S 341245 uuguuaauucacagaauagc 46AS 341216 3′UTR aguuugugcuauucugugaa 47 85 S 341246 uucacagaauagcacaaacu48 AS 341217 3′UTR agaauggcuuugugcuuagu 49 50 S 341247acuaagcacaaagccauucu 50 AS 341218 3′UTR uccaccugaaguucaccccg 51 71 S341218 cggggugaacuucaggugga 52 AS 341219 3′UTR aaggaguaucugccagacgc 5365 S 341249 gcgucuggcagauacuccuu 54 AS 341220 3′UTR uaaucacacagcaguggcaa55 72 S 341250 uugccacugcugugugauua 56 AS 341221 3′UTRgugccccgcggcucacuggg 57 34 S 341251 cccagugagccgcggggcac 58 AS 3412223′UTR aaaggauuuaggccacugcc 59 69 S 341252 ggcaguggccuaaauccuuu 60 AS341223 3′UTR acagcaucgagccaagucau 61 59 S 341253 augacuuggcucgaugcugu 62AS 341224 3′UTR acagacacacggccugcagc 63 20 S 341254 gcugcaggccgugugucugu64 AS 341225 3′UTR gaacgugacagaugugaagg 65 39 S 341255ccuucacaucugucacguuc 66 AS 341226 3′UTR uccaucaucuuacgccagac 67 73 S341256 gucuggcguaagaugaugga 68 AS 341227 3′UTR ggcgaaucaaauccaucauc 6959 S 341257 gaugauggauuugauucgcc 70 AS 341228 3′UTR auccacccugcagcucuaug71 46 S 341258 cauagagcugcaggguggau 72 AS 341229 3′UTRgccgagaugaccuccagagg 73 61 S 341259 ccucuggaggucaucucggc 74

All dsRNA compounds except ISIS 341203, 341209, 341221, 341224 and341225 demonstrated greater than 40% inhibition of survivin expression.These data suggest that at certain target sites, double-strandedcompounds with a 5′ phosphate display greater potency in inhibitingsurvivin expression (compare Tables 1 and 3).

Example 19 Comparison of siRNA Constructs Targeting the Same Site ofSurvivin mRNA Dose Response

In accordance with the present invention, the effects of altering thesequence of ISIS 339048 on inhibition of human survivin mRNA in HeLacells was investigated. ISIS 343867 (5′-UUUGAAAAUGUUGAUCUCC-3′: SEQ IDNO:81) is the antisense strand for a blunt-ended siRNA binding to thesame site on the survivin mRNA as ISIS 339048, the difference being thatISIS 343867 is a 19-mer compound which lacks the 5′-terminal adenineresidue of ISIS 339048. ISIS 341881 (UUUGAAAAUGUUGAUCUCCTT; SEQ IDNO:82) is the antisense strand for a canonical siRNA binding to the samesite on the survivin mRNA as ISIS 339048, the difference being that ISIS341881 contains dTdT (deoxythymidine-deoxythymidine) at the 3′-terminus(“dTdT “overhang”). ISIS 343868 has the sequence5′-GGAGAUCAACAUUUUCAAA-3′ (SEQ ID NO:83), and is the sense strandcorresponding to ISIS 343867. ISIS 341880 (SEQ ID NO: 84) is the sensestrand corresponding to ISIS 341881. The constructs are shown in Table 4with the antisense strand shown first followed by the sense strand, bothin 5′ to 3′ orientation. The sequences of the survivin siRNA constructsare shown in Table 4, and the dose response results are shown in Table5.

TABLE 4 siRNA constructs tested in dose response experiment in HeLacells for inhibition of human survivin mRNA expression SEQ ID ISIS #REGION SEQUENCE NO 339048 3′UTR auuugaaaauguugaucucc 23 339078ggagaucaacauuuucaaau 24 343867 3′UTR uuugaaaauguugaucucc 81 343868ggagaucaacauuuucaaa 83 341881 3′UTR uuugaaaauguugaucucctt 82 341880ggagaucaacauuuucaaatt 84

TABLE 5 Inhibition of human survivin mRNA levels by siRNA oligomericcompounds targeting the same site of survival mRNA: dose responsePercentage of Inhibition of survivin expression in HeLa cellsOligonucleotide Concentration (nM) Isis # SEQ ID: 0.02 0.08 0.4 2.0 1050 339048_339078 23_24 27 31 58 70 83 88 343867_343868 81_83 20 44 66 8290 92 341881_341880 82_84 38 52 70 81 88 91

As shown in Table 5, the compounds tested inhibit human survivin mRNAexpression in HeLa cells in a dose-dependent manner. ISIS 343867 andISIS 341881 are more effective at inhibition of survivin mRNA levels atall but the lowest doses, indicating that a blunt-ended 19-mer siRNA ora canonical 21-mer siRNA with a dTdT modification at the 3′-terminus maybe advantageous modifications of ISIS 339048, the original blunt-ended20-mer siRNA.

The IC50 values (nM) of these three compounds were as follows:

Blunt-ended siRNA (20-mer) 339048_339078, 0.28 nM; Blunt-ended siRNA(19-mer) 343867_343868, 0.19 nM and Canonical siRNA 341881_341880, 0.15nM.IC50 is defined as the concentration of oligomeric compound whichresults in 50% inhibition of mRNA (or protein) expression compared to anuntreated control. From these data, the canonical siRNA and theblunt-ended siRNA (19-mer) both performed significantly better than theblunt-ended siRNA (20-mer).

Additional blunt-ended constructs targeting human survivin: modifiedcompounds:

A series of siRNA compounds targeting human survivin (GenBank accessionno. NM_(—)01168.1; SEQ ID NO: 14) were designed and are shown in Table 6in the 5′ to 3′ orientation.

TABLE 6 siRNA compounds targeted to human survivin SEQ ID Strand ISIS#SEQUENCE NO. AS 346272 uuugaaaauguugaucuccu 85 AS 346279uuugaaaauguugaucuccuu 86 AS 346280 uuugaaaauguugaucuccu 85 AS 346281uuugaaaauguugaucucc 81 AS 346282 uuugaaaauguugaucuccuu 86 AS 346283uuugaaaauguugaucuccu 85 AS 346284 uuugaaaauguugaucucc 81 S 346286aggagaucaacauuuucaaa 87 S 346287 ggagaucaacauuuucaaa 83 S 346289aggagaucaacauuuucaaa 87 S 346290 ggagaucaacauuuucaaa 83 AS 346291uuugaaaauguugaucuccuu 86 AS 346292 uuugaaaauguugaucucc 81 S 346294aggagaucaacauuuucaaa 87 S 346295 ggagaucaacauuuucaaa 83 AS 346296uuugaaaauguugaucuccu 85 AS 348310 uuugaaaauguugaucuccuu 86 AS 352505uuugaaaauguugaucucc 81 AS 352506 uuugaaaauguugaucucc 81 AS 352507uuugaaaauguugaucucc 81 AS 352508 uuugaaaauguugaucucc 81 AS 352509uuugaaaauguugaucucc 81 AS 352510 uuugaaaauguugaucucc 81 S 352511ggagaucaacauuuucaaa 83 S 352512 ggagaucaacauuuucaaa 83 S 352513ggagaucaacauuuucaaa 83 S 352514 ggagaucaacauuuucaaa 83 AS 352515uuugaaaauguugaucucc 81 AS 353537 uuugaaaauguugaucucc 81 AS 353538uuugaaaauguugaucucc 81 AS 353539 uuugaaaauguugaucucc 81 AS 353540uuugaaaauguugaucucc 81 AS 355710 uuugaaaauguugaucucc 81 AS 355711uuugaaaauguugaucucc 81 AS 355712 uuugaaaauguugaucucc 81 AS 355713uuugaaaauguugaucucc 81 S 355714 ggagaucaacauuuucaaa 83 AS 355715uuugaaaauguugaucucc 81 AS 355716 aauuugaaaauguugaucucc 88

The modifications to the sequences in Table 6 are as follows:

ISIS 346272: all ribose, all P═O linkagesISIS 346279-346281, 346286, 346287: all P═S linkagesISIS 346282-346284, 346289 and 346290: alternating P═O/P═S linkages,beginning with P═OISIS 346291, 346292, 346294, 346295 and 346296: alternating P═S/P═Olinkage; beginning with P═SISIS 348310: all ribose with P═O backboneISIS 352505: 2′-O-methylribose at positions 5, 8, 11, 14 and 17-19. P═Obackbone.ISIS 352506: 2′-Q-methylribose at positions 6, 7, 10, II and 17-19. P═Obackbone.ISIS 352507: 2′-O-methylribose at positions 1, 3, 5, 7, 9, 11, 13, 15,17 and 19. P═O backbone.ISIS 352508: 2′-MOE at positions 5, 8, 11 and 14; 2′-O-methyl atposition 17-19. P═O backbone.ISIS 352509: 2′-MOE at positions 4, 9 and 18. P═O backbone.ISIS 352510: 2′-MOE at positions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.P═O backbone.ISIS 352511: 2′-MOE at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18. P═Obackbone.ISIS 352512: 2′-O-methyl at every position, P═O backbone.ISIS 352513: 2′-O-methyl at positions 2-18. P═O backbone.ISIS 352514: 2′-MOE at positions 2, 4, 6, 8, it, 12, 14, 16, and 18, P═Obackbone.ISIS 352515: 2′-O-methylribose at positions 15-19. PO backbone.ISIS 352516: P═S linker at linkages 1-7, P═O linker at linkages 8-18.ISIS 353537: 4′-thioribose at positions 1-3 and 17-19, P═O backbone.ISIS 353538: 4′thioribose at positions 3, 9, 12 and 17-19, P═O backbone.ISIS 353539: 4′-thioribose at positions 1-3, 9 and 12; 2′-O-methylriboseat positions 17-19, P═O backbone.ISIS 353540: 4′-thioribose at positions 1-3; 2′-O-methylribose atpositions 17-19, P═O backbone.ISIS 355710: 2′-ara-fluoro-2′-deoxyribose at positions 1-5;2′-O-methylribose at positions 15-19, P═O backbone.ISIS 355711: 2′-ara-fluoro-2′-deoxyribose at positions 1-5, 8, 9 and12-16; 2′-O-methylribose at positions 6, 7, 10, 11 and 17-19, P═Obackbone.ISIS 355712: LNA at positions 5, 8, 11, 14, 2′-O-methyl at 17-19. P═Obackbone.ISIS 355713: alternating 2′-O-methylribose/2′-ara-fluoro-2′-deoxyribose,starting with 2′OMe at position 1. P═O backbone.ISIS 355714: alternating 2′-O-methylribose/2′-ara-fluoro-2′-deoxyribose,starting with 2′-ara-fluoro at position 1. P═O backbone.ISIS 355715: LNA at positions 4, 9, 18. P═O backbone.ISIS 355716: LNA at positions 1, 2, 6, 11, 20. P═O backbone.

Example 20 Modulation of Human Survivin Expression by Single-StrandedRNAi Compounds

A series of single-stranded olgometric compounds (asRNA) was evaluatedfor their ability to inhibit human survivin in human umbilical veinendothelial cells (HUVEC). Culture methods used for HUVEC are found, forexample, at www.atcc.org.

The sequences of the asRNA oligomeric compounds are shown in Table 7.Target sites are indicated by the first (5′ most) nucleotide number, asgiven in the sequence source reference (Genbank accession no.NM_(—)001168.1, incorporated herein as SEQ ID NO: 14), to which theasRNA oligonucleotide binds.

All compounds in Table 7 are oligoribonucleotides, 20 nucleotides inlength having phosphorothioate backbone linkages throughout and aterminal phosphate on the 5p end and all are depicted in the 5′ to 3′direction.

Data were obtained by real-time quantitative PCR as described in otherexamples herein.

TABLE 7 Inhibition of human survivin mRNA levels by asRNA oligomericcompounds TARGET % SEQ ID ISIS # SITE SEQUENCE INHIB NO 347423 456auccauggcagccagcugcu 55 17 347424 512 aacccuggaaguggugcagc 69 19 347425534 aggcugguggcaccagggaa 90 21 347426 568 auuugaaaauguugaucucc 60 23347427 605 agcacaguugaaacaucuaa 54 25 347428 642 agaagcaccucuggugccac 6027 347429 756 ucccucacuucucaccuggu 62 29 347430 780 gcaaaagggacacugccuuc66 31 347431 815 aggcucugcccacgcgaaca 67 33 347432 846caacaugagguccagacaca 66 35 347433 876 aguccacacucaggacugug 60 37 347434914 uaaggaaccugcagcucaga 70 39 347435 1030 cagggacucugucuccauuc 44 41347436 1070 aacaaaauaagaaagccaug 51 43 347437 978 gcuauucugugaauuaacaa54 45 347438 1101 aguuugugcuauucugugaa 64 47 347439 1130agaauggcuuugugcuuagu 62 49 347440 1165 uccaccugaaguucaccccg 57 51 3474411212 aaggaguaucugccagacgc 66 53 347442 1232 uaaucacacagcaguggcaa 66 55347443 1258 gugccccgcggcucacuggg 54 57 347444 1305 aaaggauuuaggccacugcc60 59 347445 1329 acagcaucgagccaagucau 53 61 347446 1366acagacacacggccugcagc 52 63 347447 1394 gaacgugacagaugugaagg 47 65 3474481487 uccaucaucuuacgccagac 58 67 347449 1498 ggcgaaucaaauccaucauc 51 69347450 1529 auccacccugcagcucuaug 55 71 347451 1569 gccgagaugaccuccagagg59 73

As shown in Table 7, all asRNA compounds demonstrated at least 44%inhibition of survivin expression.

Example 21 Inhibition of Survivin mRNA Expression in HeLa cells usingdsRNA Constructs to Human Survivin

Various dsRNA constructs were tested in HeLa cells as described aboveusing the human survivin primer probe set (SEQ ID NO: 186-188) todetermine the effect of PS substitution on the ISIS 343867 19-mer (SEQID NO:81), the 339048 20-mer (SEQ ID NO: 23), and the effect of2′-O-methyl (2′-OMe), 2′-fluoro (2′-F), 2′-O-methoxyethyl (2′-MOE) and4′-thio (4′-S) chemistries. The results are shown in Tables 8 and 9. Thefirst ISIS number is the antisense strand, and the second ISIS number isthe sense strand.

TABLE 8 Inhibition of human survivin mRNA levels by blunt-ended siRNAalgometric compounds: effect of PS substitution on 19-mer Length- ISIS #backbone (antisense 5′-3′/ IC50 SEQ ID (antisense/sense) sense 3′-5′) %INHIB (nM) NO A 19 PO/PO 343867_343868 89 0.19 81_83 B 20 PO/PO339048_339078 77 1.8 23_24 C 19 PS/PS 346281_346287 76 2-20 81_83 D 19PS/PS-PO 346281_346295 88 2-20 81_83 E 19 PS/PO-PS 346281_346290 68 2-2081_83 F 19 PO-PS/PS 346284_346287 78 3.24 81_83 G 19 PS-PO/PS346292_346287 77 3.17 81_83 H 19 PS-PO/PS-PO 346292_346295 88 1.6 81_83I 19 PS-PO/PO-PS 346292_346290 94 0.13 81_83 J 19 PO-PS/PO-PS346284_346290 91 2.0 81_83 K 19 PO-PS/PS-PO 346284_346295 78 2.9 81_83

These data illustrate that the 19-mer blurt phosphodiester siRNA is moreeffective at inhibiting survivin expression and has a ten fold lowerIC50 than its 20-mer counterpart (compare lines A and B). Furthermore,this increased potency, as measured by IC50, is lost when the backbonelinkages are replaced by full phosphorothioate linkages. However, targetreduction is maintained (compare lines A and C).

It is also shown that re-introduction of phosphodiester linkages in theantisense strand in an alternating register results in recovery of theefficacy, as measured by lowered IC50 values, but not to the level ofthe full P═O backbone (for example, compare lines D and H).

Finally, alternating phosphodiester/phosphorothioate linkages in eachstrand when in opposing register (P═O in one strand opposite P═S in theother) had the greatest effect on IC50 values and expression levels,resulting in values that were better than the native optimal construct(compare lines I and A).

TABLE 9 Inhibition of human survivin mRNA levels by blunt-ended siRNAoligomeric compounds: effect of PS substitution on 20-mer Length- ISIS #backbone (antisense 5′-3′/ IC50 SEQ ID (antisense/sense) sense 3′-5′) %INHIB (nM) NO A 19 PO/PO 343867_343868 89 0.19 81_83 B 20 PO/PO339048_339078 77 1.8 23_24 C 20 PS/PS 346280_346286 88 1.5 85_87 D 20PS/PS-PO 346280_346294 84 1.7 85_87 E 20 PS/PO-PS 346280_346289 90 2.585_87 F 20 PO-PS/PS 346283_346286 77 3.9 85_87 G 20 PS-PO/PS346296_346286 86 1.8 85_87 H 20 PS-PO/PS-PO 346296_346294 86 0.85 85_87I 20 PS-PO/PO-PS 346296_346289 97 0.13 85_87 J 20 PO-PS/PO-PS346283_346294 86 0.53 85_87 K 20 PO-PS/PS-PO 346283_346289 86 0.83 85_87

These data suggest that, in contrast to 19-mers, blunt-ended 20-mershave greater tolerance for phosphorothioate backbone modifications inboth strands with IC50 values of 20-mer full P═S being comparable to20-mer with full P═O in both strands (compare lines B and C). However,both 20 mer constructs fail to achieve the IC50 seen with the 19-mer(compare lines B and C to line A).

Surprisingly, the presence of alternating internucleoside linkages inopposite register (P═O in one strand opposite P═S in the other) was ableto reduce the IC50 to that seen with the 19-mer native P═O construct(compare lines I and A).

Effects of Chemical Modifications to the Sugar

A series of blunt-ended siRNAs were designed to investigate the effectof sugar modifications on the ability of the double-stranded compoundsto inhibit expression of human survivin mRNA. The study was performed inHeLa cells as described in other examples herein and the in RNA levelsdetermined by RT-PCR.

The modifications to the compounds was as follows:

ISIS 352511 comprises 2′-O-methyl modifications (underlined) atpositions 2, 4, 6, 8, 10, 12, 14, 16 and 18.

ISIS 352512 comprises 2′-O-methyl modifications (underlined) at every2′site.

ISIS 355714 comprises 2′-fluoro modifications tin BOLD) at positions 1,3, 5, 7, 9, 11, 13, 15, 17 and 19; and 2′-O-methyl modifications atpositions 2, 4, 6, 8, 10, 12, 14, 16 and 18.ISIS 352514 comprises alternating 2′-MOE modifications positions 2, 4,6, 8, 10, 12, 14, 16 and 18.

All other compounds were native RNA compounds. The results are shown inTable 10.

TABLE 10 Inhibition of human survivin mRNA levels by siRNA oligomericcompounds: effect of sugar modifications SEQ ID IC50 % Compound Sequence5′→3′ NO: (nM) INHIB 343867 uuugaaaauguugaucucc 81 0.60 86 343868ggagaucaacauuuucaaa 83 352507 uuugaaaauguugaucucc 81 0.24 89 352511ggagaucaacauuuucaaa 83 352507 uuugaaaauguugaucucc 81 0.04 83 352512ggagaucaacauuuucaaa 83 355713 uuugaaaauguugaucucc 81 0.06 92 355714 g ga g a u c a a c a u u u u c a a a 83 352506 uuugaaaauguugaucucc 81 0.1292 352514 g g a g a u c a a c a u u u u c a a a 83 352507uuugaaaauguugaucucc 81 0.21 89 352514 g g a g a u c a a c a u u u u c aa a 83 352507 uuugaaaauguugaucucc 81 0.49 86 352514 g g a g a u c a a ca u u u u c a a a 83

These data suggest that double stranded compounds containing alternatingmotifs of 2° F. and 2′OMe are optimal constructs for the inhibition ofhuman survivin expression.

Example 22 Dose Response Experiments in Hela Cells Using ssRNAConstructs to Human Survivin

In a dose-response experiment, HeLa Sells were treated with 0.02, 0.2,2.0 and 20.0 nM of the indicated dsRNA oligonucleotide mixed with 3μg/mL LIPOFECTIN per 100 nM oligonucleotide as described by otherexamples herein. Untreated cells served as controls. Following 16 hoursof treatment, RNA was prepared from cells for subsequent real-time PCRanalysis.

Human survivin mRNA expression levels were quantitated by real-time PCRand gene target quantities were normalized using Ribogreen as describedin other examples herein. Data are averages from two experiments areshown in Table 11. The Isis number of the antisense strand is shownfirst, followed by the Isis number of the sense strand(antisense_sense).

TABLE 11 Inhibition of human survivin mRNA levels by dsRNA oligomericcompounds: dose response Percentage of Inhibition of survivin expressionin HeLa cells Oligonucleotide Concentration Seq ID 0.02 0.2 2.0 20.0Isis # Nos nM nM nM nM 343867_343868 81_83 0 54 14 84 355710_34386881_83 17 72 86 71 355711_343868 81_83 29 51 20 93 355712_343868 81_83 3863 61 92 355715_343868 81_83 40 72 75 84 355716_343868 88_83 0 18 0 41355713_355714 81_83 23 69 86 96 346280_352511 85_83 0 0 54 77346280_352512 85_83 0 13 56 81 352507_346287 81_83 0 31 74 89352505_352511 81_83 5 40 65 80 352505_352513 81_83 22 41 60 81352507_352513 81_83 17 30 67 86 339048_339078 23_24 N.D. 27 58 78339048_346286 23_87 N.D. 29 67 82 339048_346289 23_87 N.D. 18 55 82339048_346294 23_87 N.D. 39 65 85 346280_339078 85_24 N.D. 2 39 71346283_339078 85_24 N.D. 41 70 83 346296_339078 85_24 N.D. 38 69 86343867_343868 81_83 19 60 78 90 353537_343868 81_83 25 49 68 81353538_343868 81_83 17 41 72 82 353539_343868 81_83 19 48 76 88353540_343868 81_83 21 51 77 90

As shown in Table 11, many of the dsRNA compounds inhibited humansurvivin mRNA expression in HeLa cells in a dose-dependent manner.

Example 23 Dose Response Experiments in HeLa Cells Using dsRNAConstructs to Human Survivin

In a dose-response experiment, HeLa cells were treated with 0.014, 0.04,0.12, 0.37, 1.11, 3.33, 10 and 30 nM of the indicated dsRNAoligonucleotide mixed with 3 μg/mL LIPOFECTIN per 100 mM oligonucleotideas described by other examples herein. Untreated cells served ascontrols. Following 16 hours of treatment, RNA was prepared from cellsfor subsequent real-time PCR analysis.

Human survivin mRNA expression levels were quantitated by real-time PCRand gene target quantities were normalized using Ribogreen as describedin other examples herein. Data are averages from two experiments areshown in Table 12. The Isis number of the antisense strand is shownfirst, followed by the Isis number of the sense strand(antisense_sense).

TABLE 12 Inhibition of human survivin mRNA levels by dsRNA oligomericcompounds: dose response Percentage of Inhibition of survivin expressionin HeLa cells Oligonucleotide Concentration Seq ID 0.014 0.04 0.12 0.371.11 3.33 10 30 Isis # No. nM nM nM nM nM nM nM nM 353537_343868 81_83 00 0 15 17 48 77 82 353538_343868 81_83 3 25 22 37 43 70 83 82353539_343868 81_83 0 20 10 38 72 80 90 91 352507_352511 81_83 12 45 2530 29 59 78 80 352507_352512 81_83 0 45 8 25 57 65 73 83 353540_34386881_83 0 22 0 24 60 59 82 81 352506_352514 81_83 22 25 25 42 45 80 88 90352507_352514 81_83 14 12 10 41 57 77 84 90 355713_355714 81_83 30 23 4056 65 87 91 92

As shown in Table 12, most of the dsRNA compounds inhibited humansurvivin mRNA expression in HeLa cells in a dose-dependent manner.

Example 24 Double-Dependent Inhibition of Survivin mRNA Expression inU-87 MG Cells

Double-stranded compounds were tested for their ability to inhibitexpression of human survivin mRNA in U-87 MG cells using the methodsdescribed above. Various dsRNA constructs targeting human survivin weretested at concentrations of 0.0019 nM, 0.0096 nM, 0.048 nM, 0.24 nM, 1.2nM, 6.0 nM, 30.0 nM and 150.0 nM. The results are summarized in Table13. The Isis number of the antisense strand is shown first, followed bythe Isis number of the sense strand (antisense_sense).

TABLE 13 Dose dependent inhibition of human survivin mRNA expressionwith dsRNA compounds Percentage of Inhibition of survivin expression inU-87 MG cells Seq ID Oligonucleotide Concentration (nM) Isis # No.0.0019 0.0096 0.048 0.24 1.2 6.0 30 150 352506_352514 81_83 7 9 15 40 6074 82 85 352507_352514 81_83 0 0 10 30 47 65 79 84 355713_355714 81_8321 17 34 48 70 82 85 86 353537_343868 81_83 7 12 9 22 44 75 69 77353538_343868 81_83 8 10 16 25 50 65 77 79 353539_343868 81_83 5 12 1725 57 65 80 82 352507_352511 81_83 12 15 22 28 60 71 83 90 352507_35251281_83 17 3 18 27 52 66 82 85 353540_343868 81_83 16 22 33 44 58 73 83 86343867_343868 81_83 15 10 20 25 46 68 78 83 346280_346286 85_87 2 0 0 420 45 75 70 346296_346289 85_87 6 2 1 17 35 62 82 85

As shown in Table 13, most of the dsRNA compounds inhibited humansurvivin mRNA expression in U-87 MG cells in a dose-dependent manner.

Example 25 Inhibition of Survivin by Canonical siRNA Oligonucleotides

A series of canonical siRNAs were designed to target human survivin.Each of the dsRNA sequences specific to survivin and depicted belowcontain two deoxythymidine nucleotides at the 3′ terminal end of eachstrand of the RNA oligonucleotide duplex (not shown). Synthesis, duplexformation and purification of gene-specific siRNAs were performed byDharmacon Research Inc. In the Table, “Position” refers to the positionof the gene to which the antisense strand of the dsRNA binds. Eachsequence in the table is listed such that the antisense strand (topstrand) is written in 5′ to 3′ direction; its complementary sense strand(bottom strand) is also written in the 5′ to 3′ direction.

TABLE 14 Canonical siRNA oligonucleotides designed to target to humansurvivin SEQ ID Compound Strand NO Position Sequence (5′→3′) U1 AS 89015-033 ucgcgggacccguuggcag S 90 cugccaacgggucccgcga U2 AS 91 094-112ggaccaccgcaucucuaca S 92 uguagagaugcgguggucc U3 AS 93 121-139cuggcccuucuuggagggc S 94 gcccuccaagaagggccag U4 AS 95 173-191ggcuucauccacugcccca S 96 uggggcaguggaugaagcc U5 AS 97 199-217cgagccagacuuggcccag S 98 cugggccaagucuggcucg U6 AS 99 235-253ggagcuggaaggcugggag S 100 cucccagccuuccagcucc U7 AS 101 283-301aaagcauucguccgguugc S 102 gcaaccggacgaaugcuuu U8 AS 103 278-296cauaaaaagcauucguccg S 104 cggacgaaugcuuuuuaug U9 AS 105 332-350gaauuaacccuuggugaau S 106 auucaccaaggguuaauuc U10 AS 107 358-376acuggacagagaaagagcc S 108 ggcucuuucucuguccagu U11 AS 109 368-386gaaagagccaagaacaaaa S 110 uuuuguucuuggcucuuuc U12 AS 111 372-390gagccaagaacaaaauugc S 112 gcaauuuuguucuuggcuc U13 AS 113 387-405uugcaaaggaaaccaacaa S 114 uuguugguuuccuuugcaa U14 AS 115 399-417ccaacaauaagaagaaaga S 116 ucuuucuucuuauuguugg U15 AS 117 409-427gaagaaagaauuugaggaa S 118 uuccucaaauucuuucuuc U16 AS 119 429-447cugcgaagaaagugcgccg S 120 cggcgcacuuucuucgcag U17 AS 121 586-604ggagaucaacauuuucaaa S 122 uuugaaaauguugaucucc U18 AS 123 618-636cugugcuccuguuuugucu S 124 agacaaaacaggagcacag U19 AS 125 642-660guggcaccagaggugcuuc S 126 gaagcaccucuggugccac U20 AS 127 780-798gaaggcagugucccuuuug S 128 caaaagggacacugccuuc U21 AS 129 1005-1023gaugcaugacuugugugug S 130 cacacacaagucaugcauc U22 AS 131 1033-1051uggagacagagucccuggc S 132 gccagggacucugucucca U23 AS 133 1070-1088cauggcuuucuuauuuugu S 134 acaaaauaagaaagccaug U24 AS 135 1094-1112uuguuaauucacagaauag S 136 cuauucugugaauuaacaa U25 AS 137 1119-1137cuacaauuaaaacuaagca S 138 ugcuuaguuuuaauuguag U26 AS 139 1030-1048acuaagcacaaagccauuc S 140 gaauggcuuugugcuuagu U27 AS 141 1198-1216uagagugauaggaagcguc S 142 gacgcuuccuaucacucua U28 AS 143 1212-1230gcgucuggcagauacuccu S 144 aggaguaucugccagacgc U29 AS 145 1303-1321aaggcaguggccuaaaucc S 146 ggauuuaggccacugccuu U30 AS 147 1305-1323ggcaguggccuaaauccuu S 148 aaggauuuaggccacugcc U31 AS 149 1329-1347augacuuggcucgaugcug S 150 cagcaucgagccaagucau U32 AS 151 1394-1412ccuucacaucugucacguu S 152 aacgugacagaugugaagg U33 AS 153 1101-1119uucacagaauagcacaaac S 154 guuugugcuauucugugaa U34 AS 155 143-121gccugcaccccggagcgga S 156 uccgcuccggggugcaggc U35 AS 157 078-096cauaaaaagcauucguccg S 158 cggacgaaugcuuuuuaug U36 AS 159 456-474agcagcuggcugccaugga S 160 uccauggcagccagcugcu U37 AS 161 512-530gcugcaccacuuccagggu S 162 acccuggcuguggugcagc U38 AS 163 534-552uucccuggugccaccagcc S 164 ggcugguggcaccagggaa U39 AS 165 559-577ugggccccuuagcaauguc S 166 gacauugcuaaggggccca U40 AS 167 580-598aggaaaggagaucaacauu S 168 aauguugaucuccuuuccu U41 AS 169 605-623uuagauguuucaacugugc S 170 gcacaguugaaacaucuaa U42 AS 171 688-706caguggcugcuucucucuc S 172 gagagagaagcagccacug U43 AS 173 726-744cucauuuuugcuguuuuga S 174 ucaaaacagcaaaaaugag U44 AS 175 815-833uguucgcgugggcagagcc S 176 ggcucugcccacgcgaaca U45 AS 177 846-864ugugucuggaccucauguu S 178 aacaugagguccagacaca U46 AS 179 876-894cacaguccugaguguggac S 180 guccacacucaggacugug U47 AS 181 890-908uggacuuggcaggugccug S 182 caggcaccugccaagucca U48 AS 183 914-932ucugagcugcagguuccuu S 184 aaggaaccugcagcucaga U49 AS 185 946-964gugccuccucagaggacag S 186 cuguccucugaggaggcac U50 AS 187 972-990guuguuguguuuuuuuguu S 188 aacaaaaaaacacaacaac U51 AS 189 1130-1148acuaagcacaaagccauuc S 190 gaauggcuuugugcuuagu U52 AS 191 1142-1160gccauucuaagucauuggg S 192 cccaaugacuuagaauggc U53 AS 193 1152-1170gucauuggggaaacggggu S 194 accccguuuccccaaugac U54 AS 195 1165-1183cggggugaacuucaggugg S 196 ccaccuguaguucaccccg U55 AS 197 067-085gcccccugccuggcagccc S 198 gggcugccaggcagggggc U57 AS 199 1435-1453guccgcccagguccccgcu S 200 agcggggaccugggcggac U58 AS 201 1487-1505gucuggcguaagaugaugg S 202 ccaucaucuuacgccagac U59 AS 203 1498-1516gaugauggauuugauucgc S 204 gcgaaucaaauccaucauc U60 AS 205 1569-1587ccucuggaggucaucucgg S 206 ccgagaugaccuccagagg U61 AS 207 008-026auuugaaucgcgggacccg S 208 cgggucccgcgauucaaau U62 AS 209 046-064cggcaugggugccccgacg S 210 cgucggggcacccaugccg U63 AS 211 211-229ggcccaguguuucuucugc S 212 gcagaagaaacacugggcc U64 AS 213 667-685ugcagcgggugcugcuggu S 214 accagcagcacccgcugca U65 AS 215 756-774accaggugagaagugaggg S 216 cccucacuucucaccuggu U66 AS 217 1232-1250uugccacugcugugugauu S 218 aaucacacagcaguggcaa U67 AS 219 1281-1299cuggccgcuccucccucag S 220 cugagggaggagcggccag U68 AS 221 1258-1276cccagugagccgcggggca S 222 ugccccgcggcucacuggg U69 AS 223 1366-1384gcugcaggccgugugucug S 224 cagacacacggccugcagc U70 AS 225 1529-1547cauagagcugcagggugga S 226 uccacccugcagcucuaug U71 AS 227 1547-1565auuguuacagcuucgcugg S 228 ccagcgaagcuguaacaau U72 AS 229 1597-1615agaaauaaaaagccuguca S 230 ugacaggcuuuuuauuucu

Compounds U17, U20, U23, U36, U48, and U54, when tested for inhibitionof survivin mRNA using the above-described RT-PCR and quantitativeWestern blot assays, exhibit an IC₅₀ of less than 100 nM.

Compound U17 exhibits potent activity in both quantitative RT-PCRanalysis and Western blot analysis, and is therefore especiallypreferred for the indications described herein.

Example 26 IC50 values of additional double-stranded compounds targetinghuman survivin

Tables 15 and 16 summarize IC50 values using various dsRNAs which wereperformed using the methods described herein. The constructs testedcomprise the antisense stand in the 5′-3′ orientation, and the sensestrand in the 5′ to 3′ orientation. In Table 15, “D” is dose response(mRNA levels) and “W” is Western blot. All internucleoside linkages arephosphodiester unless otherwise noted by a lowercase “s” indicating aphosphorothioate linkage.

TABLE 15 IC50 data for dsRNA constructs targeted to human survivinAntisense Strand siRNA (5′-3′) Assay/ SEQ Construct Sense Strand cell ID(antisense_sense) (5′-3′) line IC50 NOs 339044_339074AGGGCUGCCAGGCAGGGGGC W/HeLa 5.3 15_16 GCCCCCUGCCUGGCAGCCCU 339045_339075AUCCAUGGCAGCCAGCUGCU W/HeLa 5.5 17_18 AGCAGCUGGCUGCCAUGGAU 339045_339075AUCCAUGGCAGCCAGCUGCU D/U-87 .68 17_18 AGCAGCUGGCUGCCAUGGAU MG339045_339075 AUCCAUGGCAGCCAGCUGCU D/HeLa 3.3 17_18 AGCAGCUGGCUGCCAUGGAU339046_339076 AACCCUGGAAGUGGUGCAGC W/HeLa 3.5 19_20 GCUGCACCACUUCCAGGGUU339047_339077 AGGCUGGUGGCACCAGGGAA W/HeLa 99.2 21_22UUCCCUGGUGCCACCAGCCU 339048_339078 AUUUGAAAAUGUUGAUCUCC W/HeLa 3.9723_24 GGAGAUCAACAUUUUCAAAU 339048_339078 AUUUGAAAAUGUUGAUCUCC D/HeLa0.42 23_24 GGAGAUCAACAUUUUCAAAU 339048_339078 AUUUGAAAAUGUUGAUCUCCD/HeLa 0.28 23_24 GGAGAUCAACAUUUUCAAAU 339048_339078AUUUGAAAAUGUUGAUCUCC D/HeLa 0.28 23_24 GGAGAUCAACAUUUUCAAAU339048_339078 AUUUGAAAAUGUUGAUCUCC D/U-87 0.34 23_24GGAGAUCAACAUUUUCAAAU MG 339048_339078 AUUUGAAAAUGUUGAUCUCC D/HeLa 1.2423_24 GGAGAUCAACAUUUUCAAAU 339048_346286 AUUUGAAAAUGUUGAUCUCC D/HeLa0.40 23_40 ASGsGsAsGsAsUsCsAsAsCsAsUsUsUs UsCsAsAsA 339048_346289AUUUGAAAAUGUUGAUCUCC D/HeLa 0.96 23_46 AGsGAsGAsUCsAAsCAsUUsUUsCAs AA339048_346294 AUUUGAAAAUGUUGAUCUCC D/HeLa 0.24 23_87AsGGsAGsAUsCAsACsAUsUUsUCsA AsA 339049_339079 AGCACAGUUGAAACAUCUAAW/HeLa 6.12 25_26 UUAGAUGUUUCAACUGUGCU 339050_339080AGAAGCACCUCUGGUGCCAC W/HeLa 100 27_28 GUGGCACCAGAGGUGCUUCU 339051_339081UCCCUCACUUCUCACCUGGU W/HeLa 100 29_30 ACCAGGUGAGAAGUGAGGGA 339052_339082GCAAAAGGGACACUGCCUUC W/HeLa 100 31_32 GAAGGCAGUGUCCCUUUUGC 339052_339082GCAAAAGGGACACUGCCUUC D/U-87 1.15 31_32 GAAGGCAGUGUCCCUUUUGC MG339052_339082 GCAAAAGGGACACUGCCUUC D/HeLa 2.41 31_32GAAGGCAGUGUCCCUUUUGC 339053_339083 AGGCUCUGCCCACGCGAACA W/HeLa 99.633_34 UGUUCGCGUGGGCAGAGCCU 339054_339084 CAACAUGAGGUCCAGACACA W/HeLa 10035_36 UGUGUCUGGACCUCAUGUUG 339055_339085 AGUCCACACUCAGGACUGUG W/HeLa 10037_38 CACAGUCCUGAGUGUGGACU 339056_339086 UAAGGAACCUGCAGCUCAGA W/HeLa 7.539_40 UCUGAGCUGCAGGUUCCUUA 339056_339086 UAAGGAACCUGCAGCUCAGA D/U-870.51 39_40 UCUGAGCUGCAGGUUCCUUA MG 339056_339086 UAAGGAACCUGCAGCUCAGAD/HeLa 1.72 39_40 UCUGAGCUGCAGGUUCCUUA 339057_339087CAGGGACUCUGUCUCCAUUC W/HeLa 100 41_42 GAAUGGAGACAGAGUCCCUG 339058_339088AACAAAAUAAGAAAGCCAUG W/HeLa 7.23 43_44 CAUGGCUUUCUUAUUUUGUU339058_339088 AACAAAAUAAGAAAGCCAUG D/HeLa 2.96 43_44CAUGGCUUUCUUAUUUUGUU 339059_339089 GCUAUUCUGUGAAUUAACAA W/HeLa 6.4145_46 UUGUUAAUUCACAGAAUAGC 339060_339090 AGUUUGUGCUAUUCUGUGAA W/HeLa4.43 47_48 UUCACAGAAUAGCACAAACU 339060_339090 AGUUUGUGCUAUUCUGUGAAD/HeLa 3.38 47_48 UUCACAGAAUAGCACAAACU 339061_339091AGAAUGGCUUUGUGCUUAGU W/HeLa 100 48_50 ACUAAGCACAAAGCCAUUCU 339062_339092UCCACCUGAAGUUCACCCCG W/HeLa 65.2 51_52 CGGGGUGAACUUCAGGUGGA339062_339092 UCCACCUGAAGUUCACCCCG D/HeLa 1.80 51_52CGGGGUGAACUUCAGGUGGA 339063_339093 AAGGAGUAUCUGCCAGACGC W/HeLa 6.2 53_54GCGUCUGGCAGAUACUCCUU 339064_339094 UAAUCACACAGCAGUGGCAA W/HeLa 5.4555_56 UUGCCACUGCUGUGUGAUUA 339065_339095 GUGCCCCGCGGCUCACUGGG W/HeLa94.2 57_58 CCCAGUGAGCCGCGGGGCAC 339066_339096 AAAGGAUUUAGGCCACUGCCW/HeLa 1.72 59_60 GGCAGUGGCCUAAAUCCUUU

TABLE 16 IC50 values for dsRNA to human survivin dsRNA (antisense_sense)SEQ ID NOS: IC50 (nM) mRNA 352505_343868 81_83 0.88 352505_346287 81_832.2 352505_352512 81_83 0.39 352505_352514 81_83 1.3 352506_346287 81_830.23 352506_352511 81_83 0.11 352506_352512 81_83 0.23 352506_35251381_83 0.43 352507_346287 81_83 0.8 352515_343868 81_83 0.29353537_352512 81_83 1.5 353537_352513 81_83 1.5 353537_352514 81_83 1.6355710_343868 81_83 0.1 346280_352514 85_83 N.D. 343867_346295 81_83N.D. 346280_339078 85_24 N.D. 346280_343868 85_83 N.D. 346281_34386881_83 N.D. 339048_346286 23_87 N.D. 343867_346287 81_83 N.D.353537_343868 81_83 0.18 353538_343868 81_83 0.16 353539_343868 81_830.13 353540_343868 81_83 0.11 346284_346287 81_83 3.24 352506_35251481_83 0.12 352507_352512 81_83 0.04 352507_352514 81_83 0.21352507_352511 81_83 0.24 352506_343868 81_83 0.16 355713_355714 81_830.06

Example 27 Measurement of Antitumor Activity in a Human GlioblastomaXenograft Tumor Model

One or more of the oligomeric compounds described herein, includingdsRNA compounds, is tested for antitumor activity in an animal modelknown in the art. Two such animal models are (I) U-87MG humanglioblastoma xenograft tumor model (Kiaris H, Schally A V, Varga J L,Antagonists of growth hormone-releasing hormone inhibit the growth ofU-87MG human glioblastoma in nude mice Neoplasia. 2000 May-June;2(3):242-50), and (2) a YUSAC-2 human melanoma xenograft tumor model(Grossman D, Kim P J, Schechner J S, Altieri D C, Inhibition of melanomatumor growth in vivo by survivin targeting. Proc Natl Acad Sci USA. 2001Jan. 16; 98(2):635-40). A total of 10 CD1 nu/nu (Charles River) mice isused for each group. For implantation, tumor cells are trypsinized,washed in PBS and resuspended in PBS at 6×10⁷ cells/ml (U-87MG) and at4×10⁷ cells/ml (YUSAC-2) in DMEM. Just before implantation, animals areirradiated (450 TBI) and cells are mixed in Matrigel (1:1). A total of6×10⁶ (U-87MG) and at 4×10⁶ (YUSAC-2) tumor cells in a 0.2 ml volume areinjected subcutaneously (s.c.) in the left rear flank. Treatment withthe test oligomeric compound (dissolved in 0.9% NaCl, injection grade),or a mismatch control oligonucleotide (dissolved in 0.9% NaCl) orvehicle (0.9% NaCl) is started 3 days post tumor cell implantation.Compounds are administered intraperitoneally (i.p.) and intravenously(i.v.) for U-87MG and YUSAC-2 studies respectively in a 0.2 ml volumeevery other day for a total of about 12 doses for the U-87MG study andabout 13 doses for the YUSAC-2 study. Tumor length and width aremeasured twice a week, and tumor volume is calculated using the formula:Tumor volume=(L×W2)×0.536. Tumor volumes are plotted against days posttumor implantation for each treatment group.

Treatment with one or more of the oligomeric compounds delays humanglioblastoma and melanoma tumor growth when compared with tumor bearinganimals treated with vehicle or 25 mg/kg mismatch controloligonucleotide.

Example 28 Stability of Alternating 2′-O-methyl/2′-fluoro siRNAConstructs in Mouse Plasma

Intact duplex RNA was analyzed from diluted mouse-plasma using anextraction and capillary electrophoresis method similar to thosepreviously described (Leeds, J. M., et al., 1996, Anal. Biochem., 235,36-43; Geary, R. S., et al., 1999, Anal. Biochem., 274, 241-248.Heparin-treated mouse plasma, from 3-6 month old female Balb/c mice(Charles River Labs) was thawed from −80° C. and diluted to 25% (v/v)with phosphate buffered saline (140 mM NaCl, 3 mM KCl, 2 mM potassiumphosphate, 10 mM sodium phosphate). Approximately 10 nmol ofpre-annealed siRNA, at a concentration of 100 μM, was added to the 25%plasma and incubated at 37° C. for 0, 15, 30, 45, 60, 120, 180, 240,360, and 420 minutes. Aliquots were removed at the indicated time,treated with EDTA to a final concentration of 2 mM, and placed on ice at0° C. until analyzed by capillary gel electrophoresis (Beckman P/ACEMDQ-UV with eCap DNA Capillary tube). The area of the siRNA duplex peakwas measured and used to calculate the percent of intact siRNAremaining. Adenosine triphosphate (ATP) was added at a concentration of2.5 mM to each injection as an internal calibration standard. A zerotime point was taken by diluting siRNA in phosphate buffered salinefollowed by capillary electrophoresis. Percent intact siRNA was plottedagainst time, allowing the calculation of a pseudo first-orderhalf-life. Results are shown in Table 18.

TABLE 17 Stability of alternating 2′-O-methyl/2′-fluoro blunt siRNAconstructs in mouse plasma Construct SEQ ID Stability (t_(1/2) in hours)353537_343868 81_83 3 355713_355714 81_83 >4ISIS 353538, the antisense strand contains 4′ thio modifications atpositions 3, 8, 11, 17-19 and is paired with a sense RNA strand which ismodified.ISIS 355713, the antisense strand contains alternating 2′Omethyl/2° F.modifications to the sugar and is paired with a sense strand havingalternating 2′F/2′Omethyl modifications. The alternate modifications arein opposing register with the antisense strand being modified with 2′Omeat position 1 while the sense strand is modified with 2° F. atposition 1. It is evident that the alternating 2′-O-methyl/2′-fluoroconstruct remains relatively unchanged and is stable in serum.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

1-71. (canceled)
 72. A compound comprising a chemically modified orunmodified doublestranded nucleic acid compound that is 19-23nucleobases in length, wherein a first strand has at least 19 contiguousnucleobases of a polynucleotide selected from the group consisting ofSEQ ID NOs: 17, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 59, 61, 65, 67, 69, 73, and 81, and wherein a second strand is100% complementary to the first strand.
 73. The compound of claim 72which is blunt-ended or canonical.
 74. The compound of claim 72comprising at least one chemical modification to a sugar, nucleobase, orinternucleoside linkage.
 75. The compound of claim 74 wherein eachchemical modification to the sugar is a 2′ modification.
 76. Thecompound of claim 75 wherein each 2′ sugar modification is independentlyselected from the group consisting of 2′-O-methoxyethyl (2′-MOE),2′-O-methyl, locked nucleic acid (LNA) and 2′-fluoro.
 77. The compoundof claim 76 wherein each 2′ modification is a 2′-O-methoxyethyl(2′-MOE).
 78. The compound of claim 76 wherein each 2′ modification is a2′-O-methyl.
 79. The compound of claim 76 wherein each 2′ modificationis a 2′-fluoro.
 80. The compound of claim 76 wherein each 2′modification of the sugar results in a bicyclic sugar.
 81. The compoundof claim 80 wherein the bicyclic modification is a locked nucleic acid(LNA).
 82. The compound of claim 74 wherein the chemical modification tothe sugar is a 4′ thio.
 83. The compound of claim 75 comprising two ormore chemically distinct 2′ sugar modifications.
 84. The compound ofclaim 74 comprising at least one internucleoside linkage modification.85. The compound of claim 84 comprising mixed phosphorothioate andphosphodiester linkages.
 86. The compound of claim 85 comprisingalternating phosphorothioate and phosphorodiester internucleosidelinkages.
 87. The compound of claim 76 comprising at least oneinternucleoside linkage modification.
 88. The compound of claim 87comprising mixed phosphorothioate and phosphodiester linkages.
 89. Thecompound of claim 88 comprising alternating phosphorothioate andphosphorodiester internucleoside linkages.
 90. The compound of claim 72comprising a conjugate.
 91. The compound of claim 72 wherein the firststrand consists of SEQ ID NO:81.
 92. The compound of claim 91 whereinthe compound is canonical.
 93. A pharmaceutical composition comprisingthe compound of claim 72 and a pharmaceutically acceptable carrier ordiluent.
 94. A method for treating a condition associated with survivinexpression or overexpression comprising administering to an animal,particularly a human, an effective amount of a compound of claim
 72. 95.The method of claim 94 wherein the condition is cancer.
 96. The methodof claim 95 wherein the cancer is selected from the group consisting ofhepatocellular cancer, breast cancer, colon cancer, prostate cancer,lung cancer, bladder cancer, ovarian cancer, renal cancer, glioblastoma,pancreatic cancer and non-Hodgkin's lymphoma.